Differential and Common Signatures of miRNA Expression and Methylation in Childhood Central Nervous System Malignancies: An Experimental and Computational Approach

© 2021 by the authors. This is an open access article distributed under the Creative Commons Attribution License. https://creativecommons.org/licenses/by/4.0/Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes’ promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study.Peer reviewe

Evidence for shared genetic risk factors between lymphangioleiomyomatosis and pulmonary function

Lymphangioleiomyomatosis; Risk factors; Pulmonary functionLinfangioleiomiomatosis; Factores de riesgo; Función pulmonarLimfangioleiomiomatosi; Factors de risc; Funció pulmonarIntroduction Lymphangioleiomyomatosis (LAM) is a rare low-grade metastasising disease characterised by cystic lung destruction. The genetic basis of LAM remains incompletely determined, and the disease cell-of-origin is uncertain. We analysed the possibility of a shared genetic basis between LAM and cancer, and LAM and pulmonary function. Methods The results of genome-wide association studies of LAM, 17 cancer types and spirometry measures (forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio and peak expiratory flow (PEF)) were analysed for genetic correlations, shared genetic variants and causality. Genomic and transcriptomic data were examined, and immunodetection assays were performed to evaluate pleiotropic genes. Results There were no significant overall genetic correlations between LAM and cancer, but LAM correlated negatively with FVC and PEF, and a trend in the same direction was observed for FEV1. 22 shared genetic variants were uncovered between LAM and pulmonary function, while seven shared variants were identified between LAM and cancer. The LAM-pulmonary function shared genetics identified four pleiotropic genes previously recognised in LAM single-cell transcriptomes: ADAM12, BNC2, NR2F2 and SP5. We had previously associated NR2F2 variants with LAM, and we identified its functional partner NR3C1 as another pleotropic factor. NR3C1 expression was confirmed in LAM lung lesions. Another candidate pleiotropic factor, CNTN2, was found more abundant in plasma of LAM patients than that of healthy women. Conclusions This study suggests the existence of a common genetic aetiology between LAM and pulmonary function

Detection of submicroscopic chromosomal rearrangements in patients with Intellectual Disability of unknown aetiology with the method of microarray comparative genomic hybridization (array-CGH)

Intellectual Disability (ID) is a very clinically and genetically heterogeneous neurodevelopmental entity, thus raising difficulties on genetic evaluation and diagnosis. Hitherto, only a few cases are detected by conventional cytogenetic and molecular techniques, while most cases remain undiagnosed and require further investigation.The present study focuses on the clinical application of array-CGH for first time in Greece as a first-tier diagnostic tool for the investigation of genetic basis of unknown etiology ID in order to identify novel pathogenic copy number variations (CNVs) and causal ID genes. All patients were referred for genetic testing by paediatricians, pediatric neurologists and auxologists from various pediatric hospitals in the whole country and/or by private doctors. Overall, 235 patients with variable degrees of ID underwent array-CGH analysis in the Department of Medical Genetics, University of Athens. They had previously undergone a series of other genetic tests (e.g conventional karyotype, X-fra, PWS/AS, RETT etc) which were negative. High resolution 4x180K and 1x244K Agilent oligo-arrays (>170.000 and >236.000 probes respectively, average resolution ~13Kb) were used in this study. On certain cases for the comprehensive evaluation and validation of molecular findings, array-CGH results were confirmed with MLPA or/and were further investigated with immunocytochemistry/immunohistochemistry, methylation-specific PCR, single gene sequencing, or/and microsatellite fragment analysis. 128 clinically significant submicroscopic aberrations were detected in 89/235 patients (57 males, 32 females, mean age 6.25 years) (~38%), explaining partially or totally the pathological phenotype. Pathogenic CNVs involve recurrent and unique genomic rearrangements (deletions and duplications) ranging in size from 0.015 to 18.4Mb at well-known and new susceptibility chromosomal loci (such as 1q44, 2q14.2, 3p25.3, 7p23.2, 7q11.23, 8q24.3, 10q26.3, 22q11.21, Xp22.31 etc). Among clinically relevant genes associated with ID, they are included; C3orf10, FLNA, LIMK1, STX1A, GTF2I, MBD5, BAI3, HERC2P4, PPP4C, CHRM5, COMT, RTN4R, MAOA/MAOB, IL1RAPL1, JARID1C, DOCK8, EHMT1, HDAC2, CYFIP1. From the total of 128 pathological CNVs that were identified and parental screening was also performed, 52 CNVs were de novo and 15 were parentally inherited. In 57 patients 1 pathological CNV was detected, whereas 32 cases had more than 1 pathogenic aberration. Moreover, 26 X-chromosome cryptic rearrangements were detected in 20 males (one familiar case, 1 mosaic). Clinically relevant CNVs involve novel pathogenic imbalances, some of them are overlapping with microdeletion/microduplication cases recorded in DECIPHER and ISCA databases or/and are related with known syndromes with atypical phenotype. Also, 7/235 patients had CNVs of unknown clinical importance (~3%), while remaining cases revealed common Copy Number Polymorphisms (CNPs)(~59%). The significant percent of positive findings might be due to the strict patient selection criteria by the expertised clinical geneticists of Department of Medical Genetics in University of Athens and high resolution microarray platform we used. Clustering and classification of more cases with similar clinical phenotype and chromosomal breakpoints will permit the identification of plausible new ID syndromes and lead to more accurate forecasting and medical interventions. Αrray-CGH is proven reliable and valid providing more precise diagnosis, better genotype-phenotype correlations as well as comprehensive genetic counselling.Η Νοητική Υστέρηση (ΝΥ) αποτελεί μια πολυπαραγοντική νευροαναπτυξιακή διαταραχή με μεγάλη κλινική και γενετική ετερογένεια που καθιστά δύσκολη τη γενετική εκτίμηση και διάγνωση. Μέχρι σήμερα, λίγες περιπτώσεις διαλευκάνονται με τις συμβατικές κυτταρογενετικές και μοριακές τεχνικές, ενώ οι περισσότερες παραμένουν αδιάγνωστες και απαιτούν περαιτέρω διερεύνηση. Η παρούσα μελέτη επικεντρώνεται στην εφαρμογή -για πρώτη φορά στην Ελλάδα- των μικροσυστοιχιών array-CGH για τη διερεύνηση του γενετικού υποβάθρου της ΝΥ αγνώστου αιτιολογίας προκειμένου να εντοπιστούν κλινικά σημαντικές ποικιλομορφίες αριθμού αντιγράφου (CNVs) και νέα παθολογικά γονίδια. Μελετήθηκαν συνολικά 235 ασθενείς με ποικίλου βαθμού ΝΥ αγνώστου αιτιολογίας με αρνητικό αποτέλεσμα από άλλες γενετικές εξετάσεις (όπως κλασικό καρυότυπο, Χ-Fra, PWS/AS, RETT ,κα) (Eργαστήριο Ιατρικής Γενετικής, ΕΚΠΑ). Όλοι οι ασθενείς παραπέμφθηκαν για γενετικό έλεγχο από παιδιάτρους, παιδονευρολόγους και αναπτυξιολόγους από διάφορα παιδιατρικά νοσοκομεία της χώρας ή/και από ιδιώτες γιατρούς. Χρησιμοποιήθηκαν μικροσυστοιχίες ολιγονουκλεοτιδίων υψηλής διακριτικής ευκρίνειας 4x180K και 1x244K της εταιρίας Agilent (>170.000 και >236.000 ανιχνευτές αντίστοιχα, μέση ευκρίνεια 13Kb). Στις περιπτώσεις που κρίθηκε απαραίτητο για τη αποσαφήνιση και ολοκληρωμένη αξιολόγηση των μοριακών ευρημάτων, τα array-CGH αποτελέσματα επιβεβαιώθηκαν με MLPA ή/και μελετήθηκαν περαιτέρω με ανοσοκυτταροχημεία/ανοσοϊστοχημεία, ειδικό για μεθυλίωση PCR, αλληλούχηση κατά Sanger ή/και ανάλυση απλοτύπων μετά από ανάλυση θραυσμάτων με χρήση μικροδορυφορικών αλληλουχιών (microsatellite fragment analysis). Ανιχνεύθηκαν 128 παθολογικά CNVs σε 89/235 ασθενείς (57 άρρενες, 32 θήλεα, μέση ηλικία 6.25 έτη) (~38%) που εξηγούν μερικώς ή ολικώς το φαινότυπο. Τα παθολογικά CNVs περιλαμβάνουν μοριακές αναδιατάξεις ελλειμμάτων και διπλασιασμών, επανεμφανιζόμενες (recurrent) και μοναδικές (unique-σε ένα μόνο ασθενή), μεγέθους από 0.015Mb έως 18.4Mb σε γνωστούς και νέους υποψήφιους γενετικούς τόπους (όπως 1q44, 2q14.2, 3p25.3, 7p23.2, 7q11.23, 8q24.3, 10q26.3, 22q11.21, Xp22.31 κα). Μεταξύ των σημαντικών γονιδίων που σχετίζονται με ΝΥ περιλαμβάνονται τα C3orf10, FLNA, LIMK1, STX1A, GTF2I, MBD5, BAI3, HERC2P4, PPP4C, CHRM5, COMT, RTN4R, MAOA/MAOB, IL1RAPL1, JARID1C, DOCK8, EHMT1, HDAC2, CYFIP1. Από τα 128 παθολογικά CNVs που εντοπίστηκαν και ελέγχθηκαν οι γονείς, 52 ήταν de novo και 15 γονεϊκής προέλευσης. Σε 57 ασθενείς εντοπίστηκε 1 παθολογικό CNV, ενώ 32 είχαν περισσότερες από 1 παθολογικές αλλαγές. Επιπλέον, ανιχνεύθηκαν 26/128 Χ-φυλοσύνδετες υπομικροσκοπικές αναδιατάξεις σε 20 άρρενες (1 οικογενής περίπτωση, 1 μωσαϊκό). Τα παθολογικά ευρήματα περιλαμβάνουν νέα CNVs, ορισμένα από τα οποία αλληλοεπικαλύπτονται με περιπτώσεις μικροελλειμμάτων/μικροδιπλασιασμών καταχωρημένες στις βάσεις δεδομένων DECIPHER και ISCA ή/και σχετίζονται με άτυπες περιπτώσεις γνωστών συνδρόμων. Επίσης, βρέθηκαν 7/235 ασθενείς με αγνώστου κλινικής σημασίας CNVs (~3%), ενώ οι υπόλοιπες περιπτώσεις αποκάλυψαν κοινές ποικιλομορφίες αριθμού αντιγράφων (Copy Number Polymorphisms, CNPs) (~59%). Το υψηλό ποσοστό των θετικών ευρημάτων μπορεί να αποδοθεί στα αυστηρά κριτήρια επιλογής των ασθενών από τους έμπειρους κλινικούς γενετιστές του Εργαστηρίου Ιατρικής Γενετικής (ΕΚΠΑ) και τη μεγάλη διακριτική ευκρίνεια των μικροσυστοιχιών που χρησιμοποιήθηκαν. Η ομαδοποίηση και ταξινόμηση περισσότερων ασθενών με παρόμοιο κλινικό φαινότυπο και χρωμοσωμικά σημεία θραύσης θα επιτρέψει την ταυτοποίηση νέων συνδρόμων ΝΥ και θα οδηγήσει σε πιο ακριβείς προγνωστικές και ιατρικές παρεμβάσεις. Οι μικροσυστοιχίες array-CGH αποδεικνύονται αξιόπιστο και έγκυρο εργαλείο πρώτης γραμμής για ακριβέστερη διάγνωση, καλύτερη συσχέτιση γονοτύπου-φαινοτύπου και ολοκληρωμένη γενετική συμβουλευτική

Differential and Common Signatures of miRNA Expression and Methylation in Childhood Central Nervous System Malignancies: An Experimental and Computational Approach

Simple Summary: Epigenetic mechanisms, that are modifications of the genome without the presence of mutations, are known to play a crucial role in central nervous system (CNS) tumors during childhood. Two well-known epigenetic regulatory mechanisms include methylation and miRNA regulatory mechanisms. Therefore, in the present study we have investigated the presence of methylated genes in childhood CNS tumors, along with miRNA expression. We have searched for correlations between gene methylation and miRNA expression. In addition, we have investigated mRNA expression in order to search for possible miRNA targets. Such approaches could prove useful for the improvement of CNS tumor prognosis, as well as for the discovery of new therapeutic targets.Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes' promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study

New miRNA profiles accurately distinguish renal cell carcinomas and upper tract urothelial carcinomas from the normal kidney.

BACKGROUND: Upper tract urothelial carcinomas (UT-UC) can invade the pelvicalyceal system making differential diagnosis of the various histologically distinct renal cell carcinoma (RCC) subtypes and UT-UC, difficult. Correct diagnosis is critical for determining appropriate surgery and post-surgical treatments. We aimed to identify microRNA (miRNA) signatures that can accurately distinguish the most prevalent RCC subtypes and UT-UC form the normal kidney. METHODS AND FINDINGS: miRNA profiling was performed on FFPE tissue sections from RCC and UT-UC and normal kidney and 434 miRNAs were significantly deregulated in cancerous vs. the normal tissue. Hierarchical clustering distinguished UT-UCs from RCCs and classified the various RCC subtypes among them. qRT-PCR validated the deregulated expression profile for the majority of the miRNAs and ROC analysis revealed their capability to discriminate between tumour and normal kidney. An independent cohort of freshly frozen RCC and UT-UC samples was used to validate the deregulated miRNAs with the best discriminatory ability (AUC>0.8, p<0.001). Many of them were located within cytogenetic regions that were previously reported to be significantly aberrated. miRNA targets were predicted using the miRWalk algorithm and ingenuity pathway analysis identified the canonical pathways and curated networks of the deregulated miRNAs. Using the miRWalk algorithm, we further identified the top anti-correlated mRNA/miRNA pairs, between the deregulated miRNAs from our study and the top co-deregulated mRNAs among 5 independent ccRCC GEO datasets. The AB8/13 undifferentiated podocyte cells were used for functional assays using luciferase reporter constructs and the developmental transcription factor TFCP2L1 was proved to be a true target of miR-489, which was the second most upregulated miRNA in ccRCC. CONCLUSIONS: We identified novel miRNAs specific for each RCC subtype and UT-UC, we investigated their putative targets, the networks and pathways in which they participate and we functionally verified the true targets of the top deregulated miRNAs

Array-CGH revealed one of the smallest 16q21q22.1 microdeletions in a female patient with psychomotor retardation

A 28-month-old girl with dysmorphic craniofacial features, microcephaly, hypotonia, psychomotor retardation, failure to thrive and gastrointestinal problems was referred for clinical evaluation. Array-CGH analysis revealed one of the smallest de novo microdeletions on chromosome 16q21q22.1, 2.03 Mb in size. Advanced molecular analysis contributes to more precise genotype-phenotype correlation and accurate definition of the breakpoints in the deleted/duplicated regions. (C) 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved

Overlapping relationship of the deregulated miRNAs.

<p>Venn diagrams illustrate the overlapping relationship of the number of up-regulated miRNAs among RCC subtypes (A), down-regulated miRNAs among RCC subtypes (B), up-regulated miRNAs among RCC subtypes and UT-UCs (C), down-regulated miRNAs between RCC subtypes and UT-UCs (D). Ninety-four miRNAs were co-upregulated among ccRCC, papRCC and chRCC; and 11, 44 and 24 miRNAs were specifically up-regulated in each one of the three RCC subtypes (ccRCC, chRCC and papRCC), respectively. On the other hand, 222 miRNAs were co-down-regulated in the three RCC subtypes, whereas 16, 18 and 5 miRNAs were specifically down-regulated in ccRCC, chRCC and papRCC, respectively. When the DE miRNAs in each RCC subtype were combined with those in UT-UC, we identified 89 and 206 miRNAs that were up- and down-regulated, respectively in all tumor types.</p

Locked Nucleic Acids-In Situ Hybridization (LNA-ISH).

<p>LNA-ISH for miR-25-5p in ccRCC (A), papRCC (B), chRCC (C) and UT-UC (D). miR-25-5p was confined to the cytoplasm both in normal and tumour sections. The nuclear expression of U6 snRNA (positive control) was confirmed in all the patient samples, whereas the scrambled oligonucleotide was negative in all samples. miR-25-5p high expression was confirmed in all ccRCC sections by LNA-ISH. ccRCCs of high stage and grade stained stronger miR-25-5p compared to lower stage and grade ccRCCs. Each ccRCC section was compared against its corresponding normal kidney section (A). papRCCs of type II stained stronger for miR-25-5p compared to type I papRCCs (B). Validating the qRT-PCR results, miR-25-5p did not stain stronger in chRCC sections vs. the normal tissue ones. This was also confirmed for chRCCs with focal sarcomatoid differentiation, suggesting that miR-25-5p does not play any role in the metastatic behavior of the tumour (C). Verifying the qRT-PCR results, miR-25-5p was not significantly stronger in UT-UC vs. the normal tissue sections (D).</p