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  • Author: D. Toncheva x
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Array-Based Comparative Genomic Hybridization Application for Revealing Genomic Micro Imbalances in Congenital Malformations

Birth defects affect 3-5% of live births and are a major cause of fetal, neonatal and infant morbidity and mortality in all industrialized countries. Some 40-60% of congenital physical anomalies in humans have no cause, 20% that seem to be multifactorial, 10-13% environmental and 12-25% genetic.

Classical cytogenetic or common comparative genomic hybridization (CGH) methods have limited use in investigation of the whole genome because of their low resolution (5-10 Mb). Fluorescence in situ hybridization (FISH) and quantitative fluorescence polymerase chain reaction (QF-PCR) have higher resolution but do not allow genome-wide screening and require some prior knowledge regarding the suspected chromosomal abnormality and its genomic location.

Because of these limitations, the impact of genetic micro imbalances as etiological factors for the development of congenital malformations (CM) is underestimated. Array-based techniques have enabled higher resolution screens for genomic imbalances in CM as they permit identification of micro aberrations with a size between 60 bp and several hundred kilobases. They make possible screening of the whole genome and detection of novel unbalanced micro structural rearrangements in a single reaction and also effective screening of new dose-dependent genes. In addition, the application of the aCGH technology has the potential to improve our understanding of the normal quantitative variants of the human genome.

Web-Based Software for Storage, Statistical Processing and Analysis of SNP Data in Studies on Complex Disorders

Single nucleotide polymorphisms (SNPs) have become a very powerful tool for molecular genetics studies. Public databases provide information on over 10 million polymorphisms in the human genome. The candidate gene approach and genomewide association studies through SNP analysis have opened a new avenue for defining the genetics of complex disorders. However, analysis of large numbers of SNPs is time-consuming, cost-intensive, and requires huge experimental and statistical resources in association studies. We have developed a web-based product that facilitates the processing and statistical analysis of SNP-genotyping data for casecontrol association studies and provides for custom design, a structured database and practical export layout. Here we describe the software product database and how it helps in high-speed comprehensive SNP analysis.

Chromatin Quality as a Crucial Factor for the Success of Fluorescent in Situ Hybridization Analyses of Unfertilized Oocytes, Polar Bodies and Arrested Zygotes

Material that is supernumerary or unsuitable for in vitro fertilization (IVF) procedures is used for basic and for IVF-related research. Despite the disadvantages of such cells, they have contributed much to our understanding of the mechanisms and prevalence of different abnormalities.

Fifty-four human unfertilized oocytes, 34 arrested bipronuclear zygotes and 15 polar bodies were fixed for analysis on the third day after in vitro insemination and were subjected to fluorescent in situ hybridization (FISH) with probes for chromosomes 18, 21, X and Y (centromere for 18, X, Y and locus-specific for 21). The aim of the study was the comparison of FISH efficiency in differently condensed chromatin.

The success of FISH analysis was over 60% of analyzed cells and it was dependent on the chromatin changes (condensation and/or fragmentation) during the culture period before cell fixation. Chromatin ageing was the crucial factor for the reduced success of FISH in both oocyte chromosomes (60.0%) and pronuclei (61.76%). The chromatin of second polar bodies (PBII), and premature chromosome condensation (PCC) of the sperm chromatin in oocytes was more suitable for FISH analysis (FISH success 75.0% in PBII and 64.29% in PCC) with both centromere and locus-specific probes.

These results revealed the significance of early signs of in vitro cell ageing for the success of FISH analysis and for the interpretation of results in case of analysis of unfertilized human ova, polar bodies and arrested zygotes.

Copy Number Changes in 1q21.3 and 1q23.3 have Different Clinical Relevance in Ovarian Tumors

Many studies have reported aberrations such as amplifications, deletions and translocations of 1q21-q23 in ovarian tumors. These findings increase the scientific interest in analyzing this region using specific gene probes. We investigated the frequency of copy number changes of two specific bacterial artificial chromosomes (BAC) clones in 1q21.3 and 1q23.3 by fluorescent in situ hybridization (FISH) on tissue microarrays consisting of 540 ovarian tumors of different malignancies, histology, stage and grade. Such changes in 1q21.3 were established in 9.64% of malignant (2.41% amplification), in 8.33% of low malignant potential (LMP) and in 13.13% of benign ovarian tumors. Copy number changes of 1q23.3 were found in 17.78% of malignant (1.48% amplification), in 16.67% of LMP and in 12.64% of benign ovarian tumors. We found a significantly higher gain of 1q23.3 in non epithelial (50%) compared to epithelial tumors (14.73%) (p <0.03). The gain of 1q21.3 prevailed in non serous malignant and LMP ovarian tumors in comparison to serous tumors. In non serous tumors, both gains were associated with higher grade. The frequency of gain in 1q23.3 was 2.5-times higher than that in 1q21.3 of ovarian cancers.

Apoptosis Gene Expression Profile in Early-Stage non Small Cell Lung Cancer

Non small cell lung cancer (NSCLC) is a highly aggressive malignancy with survival rates limited to some patients in early stages (I and II). Apoptosis resistance is a hallmark of solid tumors that is tightly concerned with their biology. We analyzed the expression of 84 apoptosis-related genes in a group of Bulgarian patients with early-stage NSCLC.

RNA samples extracted from 12 early-stage NSCLC patients [five squamous cell carcinomas (SCC) and seven adenocarcinomas (AC)] and eight adjacent non neoplastic pulmonary tissues were used for gene expression analysis. We applied pathway-focused expression profiling of 84 apoptosis-related genes using real-time PCR.

Apoptosis-related genes down regulated in NSCLC compared to non tumor lung tissue (p <0.05) included representatives of the tumor necrosis factor (TNF) ligand family [TNF superfamily 8 (TNFSF8)], caspase cascade (CASP8 and CASP10) and caspase recruitment domain (CARD) family (BCL10), the positive apoptosis regulator DAPK1 and BCL2 family member MCL1. The potential of apoptosis-related genes as prognostic and predictive markers should be validated in future studies.

Whole Genome Analysis by Array-Based Comparative Genomic Hybridization in Patients with Congenital Malformations

Congenital malformations present at delivery of an infant are due to genetic or non genetic factors and occur in 15-20% of stillborn children. Most can be diagnosed prenatally by ultrasound examination, but some can only be diagnosed after birth. Seven to 10% of infants with abnormal phenotype have numerical or structural chromosomal abnormalities that require identification for accurate diagnosis and genetic counseling. Molecular-cytogenetic and array-based techniques have enabled screening at higher resolution for congenital anomalies that result from genomic imbalances. We have examined four children with congenital anomalies, with or without mental retardation, of unclear etiology. In one child, we detected a deletion (about 28 Mb) of the region 18q21.1-18q23, in mosaic form. This abnormality was missed in a routine cytogenetic examination. We detected different polymorphic copy number variations (CNVs) in the other children. We conclude that array-based comparative genomic hybridization (CGH) is a powerful diagnostic tool for the detection of low level mosaicism.


Myelofibrosis (MF) is characterized by a presence of an extra fibrous tissue in the bone marrow and additional hematopoiesis. The somatic mutation in the Janus kinase 2 (JAK2) gene (V617F) occurs gradually and is detected in about 50.0% of myelofibrosis or essential thrombo-cytopenia (ET) patients. Our aim was to determine the genotype status according to the carriers of the V617F mutation in patients with MF at the Hematology Ward of the University Hospital "Ivan Rilski" in Sofia, Bulgaria. DNA samples were isolated from venous blood of patients with various hematological disorders. DNA was amplified by polymerase chain reaction (PCR) and subsequent restriction analysis was performed using a BsaXI restriction enzyme. The genotype status was determined on 2.0% agarose gel. We analyzed 38 patients initially suspected of carrying MF or osteomyelofibrosis (OMF). After trepanobiopsy, 20 out of 38 patients were confirmed as myelofibrotic (52.6%), 5/38 (13.2%) were diagnosed as ET, 1/38 (2.6%) was diagnosed as myeloproliferative neoplasm (MPN), 6/38 (15.8%) had polycythemia vera (PV). In six patients, the presence of disease was rejected. Patients with MF were divided into three groups according to the JAK2 V617F genotype status: homozygous for the mutation (3/20 or 15.0%), heterozygous (9/20 or 45.0%) and homozygous for the wild type allele (8/20 or 40.0%). The triggering factor of MF is still unknown. It was considered that this factor could have a genetic nature. Mutations in three genes were mainly accepted as an actual predisposing events to this disease: point mutations leading to amino acid substitutions in JAK2 (V617F) and in MPL (W515L, W515K), as well as insertion or deletion in CALK We have proven that carriers of the V617F mutation prevailed in the group of patients with MF (altogether 12 patients or 60.0%). Previous studies also showed that JAK2 V617F is present in more than half of MF patients within their blood-forming cells. Therefore, the risk of evolution to MF could be associated with V617F-mutant allele burden in patients with MPN.


Autism is a neurodevelopmental disorder of unknown origin that manifests in early childhood. Autism spectrum disorders (ASDs) refer to a broader group of neurobiological conditions, pervasive developmental disorders. Despite several arguments for a strong genetic contribution, the molecular basis in most cases remains unexplained. Several studies have reported an association between ASDs and mutations in the mitochondrial DNA (mtDNA) molecule. In order to confirm these causative relationship, we screened 21 individuals with idiopathic ASDs for a number of the most common mtDNA mutations. We identified two patients with candidate mutations: m.6852G>A that produces an amino acid change of glycine to serine in the MT-CO1 gene and m.8033A>G (Ile→Val) in the MT-CO2 gene. Overall, these findings support the notion that mitochondrial mutations are associated with ASDs. Additional studies are needed to further define the role of mitochondrial defects in the pathogenesis of autism.


The high frequency (3.0-5.0%) of congenital anomalies (CA) and intellectual disabilities (IDs), make them a serious problem, responsible for a high percentage (33.0%) of neonatal mortality. The genetic cause remains unclear in 40.0% of cases. Recently, molecular karyotyping has become the most powerful method for detection of pathogenic imbalances in patients with multiple CAs and IDs. This method is with high resolution and gives us the opportunity to investigate and identify candidate genes that could explain the genotype-phenotype correlations. This article describes the results from analysis of 81 patients with congenital malformations (CMs), developmental delay (DD) and ID, in which we utilized the CytoChip ISCA oligo microarray, 4 × 44 k, covering the whole genome with a resolution of 70 kb. In the selected group of patients with CAs, 280 copy number variations (CNVs) have been proven, 41 were pathogenic, 118 benign and 121 of unknown clinical significance (average number of variations 3.5). In six patients with established pathogenic variations, our data revealed eight pathogenic aberrations associated with the corresponding phenotype. The interpretation of the other CNVs was made on the basis of their frequency in the investigated group, the size of the variation, content of genes in the region and the type of the CNVs (deletion or duplication).


Schizophrenia is one of the major psychiatric disorders. It is a disorder of complex inheritance, involving both heritable and environmental factors. DNA methylation is an inheritable epigenetic modification that stably alters gene expression. We reasoned that genetic modifications that are a result of environmental stimuli could also make a contribution.

We have performed 26 high-resolution genomewide methylation array analyses to determine the methylation status of 27,627 CpG islands and compared the data between patients and healthy controls. Methylation profiles of DNAs were analyzed in six pools: 220 schizophrenia patients; 220 age-matched healthy controls; 110 female schizophrenia patients; 110 age-matched healthy females; 110 male schizophrenia patients; 110 age-matched healthy males. We also investigated the methylation status of 20 individual patient DNA samples (eight females and 12 males. We found significant differences in the methylation profile between schizophrenia and control DNA pools.

We found new candidate genes that principally participate in apoptosis, synaptic transmission and nervous system development (GABRA2, LIN7B, CASP3). Methylation profiles differed between the genders. In females, the most important genes participate in apoptosis and synaptic transmission (XIAP, GABRD, OXT, KRT7), whereas in the males, the implicated genes in the molecular pathology of the disease were DHX37, MAP2K2, FNDC4 and GIPC1. Data from the individual methylation analyses confirmed, the gender-specific pools results.

Our data revealed major differences in methylation profiles between schizophrenia patients and controls and between male and female patients. The dysregulated activity of the candidate genes could play a role in schizophrenia pathogenesis.