Clinical impact of copy number variation changes in bladder cancer samples.

The aim of the present study was to detect copy number variations (CNVs) related to tumour progression and metastasis of urothelial carcinoma through whole-genome scanning. A total of 30 bladder cancer samples staged from pTa to pT4 were included in the study. DNA was extracted from freshly frozen tissue via standard phenol-chloroform extraction and CNV analysis was performed on two alternative platforms (CytoChip Oligo aCGH, 4x44K and Infinium OncoArray-500K BeadChip; Illumina, Inc.). Data were analysed with BlueFuse Multi software and Karyostudio, respectively. The results highlight the role of genomic imbalances in regions containing genes with metastatic and proliferative potential for tumour invasion. A high level of genomic instability in uroepithelial tumours was observed and a total of 524 aberrations, including 175 losses and 349 gains, were identified. The most prevalent genetic imbalances affected the following regions: 1p, 1q, 2q, 4p, 4q, 5p, 5q, 6p, 6q, 7q, 8q, 9p, 9q, 10p, 10q, 11q, 13q and 17q. High-grade tumours more frequently harboured genomic imbalances (n=227) than low-grade tumours (n=103). A total of 36 CNVs in high-grade bladder tumours were detected in chromosomes 1-5, 8-11, 14, 17, 19 and 20. Furthermore, five loss of heterozygosity variants containing 176 genes were observed in high-grade bladder cancer and may be used as potential targets for precision therapy. Revealing specific chromosomal regions related to the metastatic potential of uroepithelial tumours may lay a foundation for implementing molecular CNV profiling of bladder tumours as part of a routine progression risk estimation strategy, thus expanding the personalized therapeutic approach.

16p11.2 Duplication Syndrome - a Case Report.

16p11.2 duplication syndrome is a rare disorder, often associated with intellectual disability, attention deficit, hyperactivity disorder, and a predisposition to epilepsy and schizophrenia. There are no specific dysmorphic features for this genetic condition, but micro-cephaly, micrognathia and hypertelorism could be present. We report a case of 16p11.2 duplication syndrome which has the typical clinical presentation - slight facial dysmorphism, impaired intellectual development, and autistic behavior. Whole-exome sequencing was performed, but no pathogenic or likely pathogenic mutations were identified. Array comparative genomic hybridization analysis established the diagnosis of 16p11.2 duplication syndrome, which illustrates the importance of this method when diagnosing children with unexplained intellectual disability.

Unbalanced human embryos secrete more hyperglycosylated human chorionic gonadotrophin (hCG-H) than balanced ones.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Centenarian Exomes as a Tool for Evaluating the Clinical Relevance of Germline Tumor Suppressor Mutations.

OBJECTIVES: The aim of the present study was to evaluate the clinical relevance of mutations in tumor suppressor genes using whole-exome sequencing data from centenarians and young healthy individuals. METHODS: Two pools, one of centenarians and one of young individuals, were constructed and whole-exome sequencing was performed. We examined the whole-exome sequencing data of Bulgarian individuals for carriership of tumor suppressor gene variants. RESULTS: Of all variants annotated in both pools, 5080 (0.06%) are variants in tumor suppressor genes but only 46 show significant difference in allele frequencies between the two studied groups. Four variants (0.004%) are pathogenic/risk factors according to single nucleotide polymorphism database: rs1566734 in PTPRJ, rs861539 in XRCC3, rs203462 in AKAP10, and rs486907 in RNASEL. DISCUSSION: Based on their high minor allele frequencies and presence in the centenarian group, we could reclassify them from pathogenic/risk factors to benign. Our study shows that centenarian exomes can be used for re-evaluating the clinically uncertain variants.

Ancient human mitochondrial genomes from Bronze Age Bulgaria: new insights into the genetic history of Thracians.

One of the best documented Indo-European civilizations that inhabited Bulgaria is the Thracians, who lasted for more than five millennia and whose origin and relationships with other past and present-day populations are debated among researchers. Here we report 25 new complete mitochondrial genomes of ancient individuals coming from three necropolises located in different regions of Bulgaria - Shekerdja mogila, Gabrova mogila and Bereketska mogila - dated to II-III millennium BC. The identified mtDNA haplogroup composition reflects the mitochondrial variability of Western Eurasia. In particular, within the ancient Eurasian genetic landscape, Thracians locate in an intermediate position between Early Neolithic farmers and Late Neolithic-Bronze Age steppe pastoralists, supporting the scenario that the Balkan region has been a link between Eastern Europe and the Mediterranean since the prehistoric time. Spatial Principal Component Analysis (sPCA) performed on Thracian and modern mtDNA sequences, confirms the pattern highlighted on ancient populations, overall indicating that the maternal gene pool of Thracians reflects their central geographical position at the gateway of Europe.

MicroRNA Profiling in Patients with Upper Tract Urothelial Carcinoma Associated with Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a disease that affects people that live in the alluvial plains along the tributaries of the Danube River in the Balkan region. BEN is a chronic tubulointerstitial disease with a slow progression to terminal renal failure and has strong association with upper tract urothelial carcinoma (UTUC). There are several hypotheses about the etiology of BEN, but only the toxic effect of aristolochic acid has been confirmed as a risk factor in the occurrence of the disease. Aberrantly expressed miRNAs have been shown to be associated with many types of cancers. A number of studies have investigated the expression of microRNAs in urothelial carcinoma, mainly on urothelial bladder cancer, and only a few have included patients with UTUC. Here we present the first study of microRNA profiling in UTUC tissues from patients with BEN (BEN-UTUC) and patients with UTUC from nonendemic Balkan regions (non-BEN-UTUC) in comparison to normal kidney tissues. We found 10 miRNAs that were differentially expressed in patients with BEN-UTUC and 15 miRNAs in patients with non-BEN-UTUC. miRNA signature determined in BEN-UTUC patients differs from the non-BEN-UTUC patients; only miR-205-5p was mutual in both groups.

Changes in the gene expression profile of the bladder cancer cell lines after treatment with Helix lucorum and Rapana venosa hemocyanin.

PURPOSE: The purpose of this study was to elucidate the mechanism of action of the Helix lucorum hemocyanin (HlH), b-HlH-h, and RvH2-g hemocyanins as potential agents against bladder cancer. METHODS: We evaluated the viability of 647-V, T-24, and CAL-29 bladder cancer cell lines after treatment with the tested hemocyanins. The cell viability was measured at 72 hrs with MTT and WST-1 assays. Acridine orange/propidium iodide double staining was used to discriminate between apoptotic and necrotic cells. Gene expression profiling of the 168 genes from human inflammatory cytokines and signal transduction pathways were performed on the tumor cells before and after hemocyanins' treatment. RESULTS: The results showed decreased survival of cancer cells in the presence of HlH and two functional units: b-HlH-h and RvH2-g. Acridine orange/propidium iodide double staining revealed that the decreased viability was due to apoptosis. The gene expression data showed upregulation of genes involved in the apoptosis as well as of the immune system activation, and downregulation of the CCL2, CCL17, CCL21, CXCL1, and ABCF1 genes. CONCLUSIONS: The present study is the first to report gene expression in human cells under the influence of hemocyanins. The mechanism of antitumor activity of the HlH, b-HlH-h, and RvH2-g hemocyanins includes induction of apoptosis. In addition to the antiproliferative effect, downregulation of the genes with metastatic potential was observed. Together with the already known immunogenic effect, these findings support further studies on hemocyanins as potential therapeutic agents against bladder cancer.

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Methylation analysis on whole genome level. What did we learn from BEN studies?

Recent years have brought the interest of genetic research to the human epigenome. Its unique characteristics--being simultaneously stable and dynamic, present vast opportunities for cell function regulation, maintaining and executing different cell type programmes. Greatest efforts were invested in studying DNA methylation in comparison to histone modifications and miRNA studies. The present review aims to shed some light on the key mechanisms governing the epigenetic characteristics of the human genome and how epigenetic patterns can be analysed on whole genome scale. Here are presented the main advantages and disadvantages of different approaches to methylation analysis. This review presents a link between the methodology and some of its application in BEN research as an example of polygenic disorders.

Whole genome methylation analyses of schizophrenia patients before and after treatment.

The aetiology of schizophrenia is still unknown but it involves both heritable and non-heritable factors. DNA methylation is an inheritable epigenetic modification that stably alters gene expression. It takes part in the regulation of neurodevelopment and may be a contributing factor to the pathogenesis of brain diseases. It was found that many of the antipsychotic drugs may lead to epigenetic modifications. We have performed 42 high-resolution genome-wide methylation array analyses to determine the methylation status of 27,627 CpG islands. Differentially methylated regions were studied with samples from 20 Bulgarian individuals divided in four groups according to their gender (12 males/8 females) and their treatment response (6 in complete/14 in incomplete remission). They were compared to two age and sex matched control pools (110 females in female pool/110 males in male pool) before and after treatment. We found significant differences in the methylation profiles between male schizophrenia patients with complete remission and control male pool before treatment (C16orf70, CST3, DDRGK1, FA2H, FLJ30058, MFSD2B, RFX4, UBE2J1, ZNF311) and male schizophrenia patients with complete remission and control male pool after treatment (AP1S3, C16orf59, KCNK15, LOC146336, MGC16384, XRN2) that potentially could be used as target genes for new therapeutic strategies as well as markers for good treatment response. Our data revealed major differences in methylation profiles between male schizophrenia patients in complete remission before and after treatment and healthy controls which supports the hypothesis that antipsychotic drugs may play a role in epigenetic modifications.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology.

BACKGROUND: Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. METHODS: We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. RESULTS: Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. CONCLUSION: Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.

Association of a bitter taste receptor mutation with Balkan Endemic Nephropathy (BEN).

BACKGROUND: Balkan Endemic Nephropathy (BEN) is late-onset kidney disease thought to arise from chronic exposure to aristolochic acid, a phytotoxin that contaminates wheat supplies in rural areas of Eastern Europe. It has recently been demonstrated that humans are capable of perceiving aristolochic acid at concentrations below 40 nM as the result of high-affinity interactions with the TAS2R43 bitter taste receptor. Further, TAS2R43 harbors high-frequency loss-of-function mutations resulting in 50-fold variability in perception. This suggests that genetic variation in TAS2R43 might affect susceptibility to BEN, with individuals carrying functional forms of the receptor being protected by an ability to detect tainted foods. METHODS: To determine whether genetic variation in TAS2R43 predicts BEN susceptibility, we examined genotype-phenotype associations in a case-control study. A cohort of 88 affected and 99 control subjects from western Bulgaria were genotyped with respect to two key missense variants and a polymorphic whole-gene deletion of TAS2R43 (W35S, H212R, and wt/Δ), which are known to affect taste sensitivity to aristolochic acid. Tests for association between haplotypes and BEN status were then performed. RESULTS: Three major TAS2R43 haplotypes observed in previous studies (TAS2R43-W35/H212, -S35/R212 and -Δ) were present at high frequencies (0.17, 0.36, and 0.47 respectively) in our sample, and a significant association between genotype and BEN status was present (P = 0.020; odds ratio 1.18). However, contrary to expectation, BEN was positively associated with TAS2R43-W35/H212, a highly responsive allele previously shown to confer elevated bitter sensitivity to aristolochic acid, which should drive aversion but might also affect absorption, altering toxin activation. CONCLUSIONS: Our findings are at strong odds with the prediction that carriers of functional alleles of TAS2R43 are protected from BEN by an ability to detect and avoid aristolochic acid exposure. Evidence for a positive association between high-sensitivity alleles and BEN status suggests instead that possession of toxin-responsive receptor variants may paradoxically increase vulnerability, possibly by shifting attractive responses associated with low-intensity bitter sensations. The broad-spectrum tuning of the ~25-member TAS2R family as a whole toward xenobiotics points to a potentially far-reaching relevance of bitter responses to exposure-related disease in both individuals and populations.