Dual Effect of Taxifolin on ZEB2 Cancer Signaling in HepG2 Cells.

Polyphenols, secondary metabolites of plants, exhibit different anti-cancer and cytoprotective properties such as anti-radical, anti-angiogenic, anti-inflammation, or cardioprotective. Some of these activities could be linked to modulation of miRNAs expression. MiRNAs play an important role in posttranscriptional regulation of their target genes that could be important within cell signalling or preservation of cell homeostasis, e.g., cell survival/apoptosis. We evaluated the influence of a non-toxic concentration of taxifolin and quercetin on the expression of majority human miRNAs via Affymetrix GeneChip™ miRNA 3.0 Array. For the evaluation we used two cell models corresponding to liver tissue, Hep G2 and primary human hepatocytes. The array analysis identified four miRNAs, miR-153, miR-204, miR-211, and miR-377-3p, with reduced expression after taxifolin treatment. All of these miRNAs are linked to modulation of ZEB2 expression in various models. Indeed, ZEB2 protein displayed upregulation after taxifolin treatment in a dose dependent manner. However, the modulation did not lead to epithelial mesenchymal transition. Our data show that taxifolin inhibits Akt phosphorylation, thereby diminishing ZEB2 signalling that could trigger carcinogenesis. We conclude that biological activity of taxifolin may have ambiguous or even contradictory outcomes because of non-specific effect on the cell.

Epithelial to mesenchymal transition and microRNA expression are associated with spindle and apocrine cell morphology in triple-negative breast cancer.

Triple negative breast cancers (TNBC) are a morphologically and genetically heterogeneous group of breast cancers with uncertain prediction of biological behavior and response to therapy. Epithelial to mesenchymal transition (EMT) is a dynamic process characterized by loss of typical epithelial phenotype and acquisition of mesenchymal characteristics. Aberrant activation of EMT can aggravate the prognosis of patients with cancer, however, the mechanisms of EMT and role of microRNAs (miRNAs) in EMT activation is still unclear. The aim of our study was to analyze miRNA expression within areas of TNBCs with cellular morphology that may be related to the EMT process and discuss possible associations. Out of all 3953 re-examined breast cancers, 460 breast cancers were diagnosed as TNBC (11.64%). With regard to complete tumor morphology preservation, the tissue samples obtained from core-cut biopsies and influenced by previous neoadjuvant therapy were excluded. We assembled a set of selected 25 cases to determine miRNA expression levels in relation to present focal spindle cell and apocrine cell morphology within individual TNBCs. We used descriptive (histological typing and morphology), morphometric, molecular (microdissection of tumor and non-tumor morphologies, RNA isolation and purification, microchip analysis) and bioinformatic analysis (including pathway analysis). The results were verified by quantitative real-time PCR (RT-qPCR) on an extended set of 70 TNBCs. The majority of TNBCs were represented by high-grade invasive carcinomas of no special type (NST) with medullary features characterized by well-circumscribed tumors with central necrosis or fibrosis and frequent tendency to spindle-cell and/or apocrine cell transformation. Apocrine and spindle cell transformation showed a specific miRNA expression profile in comparison to other tumor parts, in situ carcinoma or non-tumor structures, particularly down-regulated expression of hsa-miRNA-143-3p and hsa-miRNA-205-5p and up-regulated expression of hsa-miR-22-3p, hsa-miRNA-185-5p, and hsa-miR-4443. Apocrine cell tumor morphology further revealed decreased expression of hsa-miR-145-5p and increased expression of additional 14 miRNAs (e.g. hsa-miR-182-5p, hsa-miR-3135b and hsa-miR-4417). Pathway analysis for target genes of these miRNAs revealed several shared biological processes (i.e. Wnt signaling, ErbB signaling, MAPK signaling, endocytosis and axon guidance), which may in part contribute to the EMT and tumor progression. We provide the first miRNA expression profiling of specific tissue morphologies in TNBC. Our results demonstrate a specific miRNA expression profile of apocrine and spindle cell morphology which can exhibit a certain similarity with the EMT process and may also be relevant for prognosis and therapy resistance of TNBC.

Differences in the importance of microcephaly, dysmorphism, and epilepsy in the detection of pathogenic CNVs in ID and ASD patients.

BACKGROUND: Autism spectrum disorders (ASD) and intellectual disabilities (ID) are heterogeneous and complex developmental diseases with significant genetic backgrounds and overlaps of genetic susceptibility loci. Copy number variants (CNVs) are known to be frequent causes of these impairments. However, the clinical heterogeneity of both disorders causes the diagnostic efficacy of CNV analysis to be modest. This could be resolved by stratifying patients according to their clinical features. AIM: First, we sought to assess the significance of particular clinical features for the detection of pathogenic CNVs in separate groups of ID and ASD patients and determine whether and how these groups differ from each other in the significance of these variables. Second, we aimed to create a statistical model showing how particular clinical features affect the probability of pathogenic CNV findings. METHOD: We tested a cohort of 204 patients with ID (N = 90) and ASD (N = 114) for the presence of pathogenic CNVs. We stratified both groups according to their clinical features. Fisher's exact test was used to determine the significance of these variables for pathogenic CNV findings. Logistic regression was used to create a statistical model of pathogenic CNV findings. RESULTS: The frequency of pathogenic CNV was significantly higher in the ID group than in the ASD group: 18 (19.78%) versus 8 (7%) (p < 0.004). Microcephaly showed a significant association with pathogenic findings in ID patients (p < 0.01) according to Fisher's exact test, whereas epilepsy showed a significant association with pathogenic findings in ASD patients (p < 0.01). The probability of pathogenic CNV findings when epilepsy occurred in ASD patients was more than two times higher than if epilepsy co-occurred with ID (29.6%/14.0%). Facial dysmorphism was a significant variable for detecting pathogenic CNVs in both groups (ID p = 0.05, ASD p = 0.01). However, dysmorphism increased the probability of pathogenic CNV detection in the ID group nearly twofold compared to the ASD group (44.4%/23.7%). The presence of macrocephaly in the ASD group showed a 25% probability of pathogenic CNV findings by logistic regression, but this was insignificant according to Fisher's exact test. The probability of detecting pathogenic CNVs decreases up to 1% in the absence of dysmorphism, macrocephaly, and epilepsy in the ASD group. CONCLUSION: Dysmorphism, microcephaly, and epilepsy increase the probability of pathogenic CNV findings in ID and ASD patients. The significance of each feature as a predictor for pathogenic CNV detection differs depending on whether the patient has only ASD or ID. The probability of pathogenic CNV findings without dysmorphism, macrocephaly, or epilepsy in ASD patients is low. Therefore the efficacy of CNV analysis is limited in these patients.

MLPA is a practical and complementary alternative to CMA for diagnostic testing in patients with autism spectrum disorders and identifying new candidate CNVs associated with autism.

BACKGROUND: Autism spectrum disorder (ASD) is a complex heterogeneous developmental disease with a significant genetic background that is frequently caused by rare copy number variants (CNVs). Microarray-based whole-genome approaches for CNV detection are widely accepted. However, the clinical significance of most CNV is poorly understood, so results obtained using such methods are sometimes ambiguous. We therefore evaluated a targeted approach based on multiplex ligation-dependent probe amplification (MLPA) using selected probemixes to detect clinically relevant variants for diagnostic testing of ASD patients. We compare the reliability and efficiency of this test to those of chromosomal microarray analysis (CMA) and other tests available to our laboratory. In addition, we identify new candidate genes for ASD identified in a cohort of ASD-diagnosed patients. METHOD: We describe the use of MLPA, CMA, and karyotyping to detect CNV in 92 ASD patients and evaluate their clinical significance. RESULT: Pathogenic and likely pathogenic mutations were identified by CMA in eight (8.07% of the studied cohort) and 12 (13.04%) ASD patients, respectively, and in eight (8.07%) and four (4.35%) patients, respectively, by MLPA. The detected mutations include the 22q13.3 deletion, which was attributed to ring chromosome 22 formation based on karyotyping. CMA revealed a total of 91 rare CNV in 55 patients: eight pathogenic, 15 designated variants of unknown significance (VOUS)-likely pathogenic, 10 VOUS-uncertain, and 58 VOUS-likely benign or benign. MLPA revealed 18 CNV in 18 individuals: eight pathogenic, four designated as VOUS-likely pathogenic, and six designated as VOUS-likely benign/benign. Rare CNVs were detected in 17 (58.62%) out of 29 females and 38 (60.32%) out of 63 males in the cohort. Two genes, DOCK8 and PARK2, were found to be overlapped by CNV designated pathogenic, VOUS-likely pathogenic, or VOUS-uncertain in multiple patients. Moreover, the studied ASD cohort exhibited significant (p < 0.05) enrichment of duplications encompassing DOCK8. CONCLUSION: Multiplex ligation-dependent probe amplification and CMA yielded concordant results for 12 patients bearing CNV designated pathogenic or VOUS-likely pathogenic. Unambiguous diagnoses were achieved for eight patients (corresponding to 8.7% of the total studied population) by both MLPA and CMA, for one (1.09%) patient by karyotyping, and for one (1.09%) patient by FRAXA testing. MLPA and CMA thus achieved identical reliability with respect to clinically relevant findings. As such, MLPA could be useful as a fast and inexpensive test in patients with syndromic autism. The detection rate of potentially pathogenic variants (VOUS-likely pathogenic) achieved by CMA was higher than that for MLPA (13.04% vs. 4.35%). However, there was no corresponding difference in the rate of unambiguous diagnoses of ASD patients. In addition, the results obtained suggest that DOCK8 may play a role in the etiology of ASD.

Haploinsufficiency of BMP4 and OTX2 in the Foetus with an abnormal facial profile detected in the first trimester of pregnancy.

BACKGROUND: Interstitial microdeletion 14q22q23 is a rare chromosomal syndrome associated with variable defects: microphthalmia/anophthalmia, pituitary anomalies, polydactyly/syndactyly of hands and feet, micrognathia/retrognathia. The reports of the microdeletion 14q22q23 detected in the prenatal stages are limited and the range of clinical features reveals a quite high variability. CASE PRESENTATION: We report a detection of the microdeletion 14q22.1q23.1 spanning 7,7 Mb and involving the genes BMP4 and OTX2 in the foetus by multiplex ligation-dependent probe amplification (MLPA) and verified by microarray subsequently. The pregnancy was referred to the genetic counselling for abnormal facial profile observed in the first trimester ultrasound scan and micrognathia (suspicion of Pierre Robin sequence), hypoplasia nasal bone and polydactyly in the second trimester ultrasound scan. The pregnancy was terminated on request of the parents. CONCLUSION: An abnormal facial profile detected on prenatal scan can provide a clue to the presence of rare chromosomal abnormalities in the first trimester of pregnancy despite the normal result of the first trimester screening test. The patients should be provided with genetic counselling. Usage of quick and sensitive methods (MLPA, microarray) is preferable for discovering a causal aberration because some of the CNVs cannot be detected with conventional karyotyping in these cases. To the best of our knowledge, this is the earliest detection of this microdeletion (occurred de novo), the first case detected by MLPA and confirmed by microarray. Literature review of the genotype-phenotype correlation in similar reports leads us to the conclusion that dosage imbalance of the chromosomal segment 14q22q23 (especially haploinsuffiency of the genes BMP4 and OTX2) contributes significantly to orofacial abnormalities. Association of the region with the Pierre Robin sequence appears to be plausible.