[SECOND STAGE IN MINIINVASIVE FETAL SURGERY FOR SEVERE CONGENITAL DIAPHRAGMATIC HERNIA. CASE REPORT].

We present a case of miniinvasive fetal surgery for CDH treated at 28 and 34 weeks of gestation. The first step was successfully performed at 28 weeks with Fetal Endoscopic Tracheal Occlusion with ballon. The second step was performed at 34 weeks for balloon removal. The necessity of fetal cytogenetic assessment and array CGH was carried out to exclude gene disorders that could lead to poor long-term outcome. A planned SC and optimal neonatology management were followed by a surgical operation of the newborn. Experienced interdisciplinary team successfully provide a perinatal and postnatal surgery for severe CDH. The newborn was discharged from the hospital 3 weeks after the repairing operation in a good condition.

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.

Epigenetic alterations in patients with type 2 diabetes mellitus.

Epigenetic changes, in particular DNA methylation processes, play a role in the pathogenesis and progression of type 2 diabetes mellitus (T2DM) linking genetic and environmental factors. To clarify this role, we have analyzed in patients with different duration of T2DM: (i) expression levels of methyl-CpG-binding domain protein 2 (MBD2) as marker of DNA methylation, and ii) methylation changes in 22 genes connected to cellular stress and toxicity. We have analyzed MBD2 mRNA expression levels in16 patients and 12 controls and the methylation status of stress and toxicity genes in four DNA pools: (i) controls; (ii) newly-diagnosed T2DM patients; (iii) patients with T2DM duration of <5 years and (iv) of >5 years. The MBD2 expression levels were 10.4-times increased on average in T2DM patients compared to controls. Consistent increase in DNA methylation fraction with the increase in T2DM duration was observed in Prdx2 and SCARA3 genes, connected to oxidative stress protection and in BRCA1 and Tp53 tumor-suppressor genes. In conclusion, increased MBD2 expression in patients indicated general dysregulation of DNA methylation in T2DM. The elevated methylation of Prdx2 and SCARA3 genes suggests disturbance in oxidative stress protection in T2DM. The increased methylation of BRCA1 and Tp53 genes unraveled an epigenetic cause for T2DM related increase in cancer risk.

Peutz-Jeghers syndrome--a rare case and a literature review.

The Peutz-Jeghers syndrome is inherited condition, characterized by hamartomatous gastrointestinal polyposis and with mucocutaneous pigmentation. We have experienced a case with typical clinical features, diagnosed before complication's development. In order to prevent cancer setting it is recommended to perform aggressive screening and high-technological procedures.

NGS nominated CELA1, HSPG2, and KCNK5 as candidate genes for predisposition to Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants--CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients 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.

Genome-wide association study on bipolar disorder in the Bulgarian population.

Bipolar disorder is a severe psychiatric disorder influenced by environmental and genetic factors. Genetic studies have implicated many variants in the disease's etiology but only few have been successfully replicated. We conducted a genome-wide association study (GWAS) on bipolar disorder in the Bulgarian population followed by a replication study of the top 100 single nucleotide polymorphisms (SNPs) showing the smallest P values. The GWAS was performed on 188 bipolar disorder patients and 376 control subjects genotyped on the Illumina 550 platform. The replication study was conducted on 122 patients and 328 controls. Although our study did not show any association P value that achieved genome-wide significance, and none of the top 100 SNPs reached the Bonferroni-corrected P value in the replication study, the plausible involvement of some variants cannot be entirely discarded. Three polymorphisms, rs8099939 [P = 2.12 × 10(-6), odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.43-2.67] in GRIK5, rs6122972 (P = 3.11 × 10(-6), OR = 2.02, 95% CI = 1.46-2.80) in PARD6B and rs2289700 (P = 9.14 × 10(-6), OR = 2.13, 95% CI = 1.53-2.95) in CTSH remained associated at a similar level after Mantel-Haenszel test for combining the results from the genome-wide and replication studies. A modest association was also detected for SNP rs1012053 (GWAS P = 4.50 × 10(-2)) in DGKH, which has already been reported as the most significant variant in a previous genome-wide scan on bipolar disorder. However, further studies using larger datasets are needed to identify variants with smaller effects that contribute to the risk of bipolar disorder.

Amplification of c-MYC and MLL Genes as a Marker of Clonal Cell Progression in Patients with Myeloid Malignancy and Trisomy of Chromosomes 8 or 11.

Gene amplification (amp) is one of the basic mechanisms connected with overexpression of oncogenes. The c-MYC (located in 8q24) and MLL (located in 11q23) are the most often over represented genes that lead to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Assessment of the level of amp c-MYC or amp MLL in the cases with trisomy 8 (+8) or trisomy 11 (+11) and myeloid malignances is necessary for a more precise estimation of the disease progression. A total of 26 patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) were included in the study: 18 with +8, six with +11 and two with complex karyotypes suspected of the partial trisomy. Routine cytogenetic analysis and fluorescent in situ hybridization (FISH) were applied to indicate the chromosome alterations and genes amp in the bone marrow cells. Amp c-MYC was observed in 12 from 18 (66.7%) patients with +8. All the patients with +11 demonstrated a different level of amp MLL. In most of the cases with MDS (9/10), the coincidence of the +8 or +11 with amp c-MYC or amp MLL, respectively, leads to transformation to AML and/or short overall survival. Our data suggest that amp c-MYC and amp MLL develop in conformity with +8 and +11, especially in cases with progressive deviations in the karyotype as an aggressive expansion of an aberrant cell clone and appearance of additional chromosome anomalies.

[Clinical, histopathological and cytogenetical findings in six cases with primary glioblastoma multiforme].

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. There is an increasing amount of data demonstrating that the routine histological examination has limited value to predict the tumor biological behaviour. AIM: To compare the clinical, histological and cytogenetical findings in a group of six patients with primary GBM surgically treated in the Department of Neurosurgery at University Hospital "St. Ivan Rilski"- Sofia, Bulgaria. MATERIAL AND METHODS: The studied group consisted of three women and three men with average age of 51 years and 6 months. In all patients the diagnosis was histologically confirmed. A microarray comparative genomic hybridization (CGH) analysis of fresh-frozen tumor tissue samples was also made. RESULTS: In two of the patients the tumor was localized in frontotemporal region, in another two- in frontoparietal, and in the other two- in parietal and occipital respectively. The onset was with headache in three of the cases. The median time between the onset and admission in the clinic was 70 days. Gross-total tumor removal was performed in one patient. In the other five subtotal excision was made. Three of the patients improved after the intervention. One patient deteriorated after the surgery. All patients demonstrated typical histological findings except one who had giant cell subtype of GBM. The microarray CGH analysis determined chromosome 10 monosomy in five patients, trisomy 7 - in four, trisomy 20 - in three, 3q23 deletion - in three, and Yp11.2 deletion - in three. CONCLUSION: GBMs are genetically heterogeneous tumors with different clinical response to standard multimodal treatment regimens. The microarray CGH analysis is a powerful method which can demonstrate the presence of a number of molecular markers with possible predictive value.

Tissue microarray analysis of EGFR and erbB2 copy number changes in ovarian tumors.

The objective of this study was to assess the implication of copy number changes of epidermal growth factor receptor (EGFR) and erbB2 genes in the etiology and progression of ovarian tumors. In our study, we used the highly reliable method of fluorescent in situ hybridization, applied on tissue microarray, containing 1006 ovarian tumors from different malignancy, histologic type and grade, and tumor stage, in order to analyze the correlations between gene copy number changes and tumor phenotype. We established copy number changes of erbB2 in 15.30% of malignant ovarian tumors-8.16% amplifications and 7.14% gains. The frequency of EGFR copy number changes was 10.67%-3.65% amplifications and 7.02% gains. EGFR gains occurred with approximately the same frequency in malignant (7.02%), low malignant potential (8.33%), and benign (7.19%) ovarian tumors. ErbB2 amplification was associated with clear cell type of ovarian cancer (P < 0.04). No amplification of EGFR and erbB2 genes was established in tumors with low malignant potency and in benign tumors. Regarding cancer phenotype, there was no statistically significant association between erbB2 copy number changes and histologic grade as well as tumor stage of ovarian cancer. EGFR gains are early events in ovarian tumorigenesis. Our results showed similar frequencies of EGFR gains in different grade tumors, while EGFR amplification increased from grades 1 to 2 to 3.

Correlations between c-myc gene copy-number and clinicopathological parameters of ovarian tumours.

The objective of this study was to investigate increases in c-myc gene copy-number in ovarian tumours, and to analyze their correlations with clinicopathological parameters. Here we applied FISH on TMA (tissue microarrays) containing 507 ovarian tumour samples from different malignancy, histology, stage and grade. Overall, we found high frequency for c-myc copy-number increases (38.5%) in ovarian cancers: 22.1% amplifications and 16.4% gains. We established c-myc amplification in more than 30% in endometrioid and mixed epithelial ovarian carcinomas. c-myc gains were found in a high proportion (42.9%) of clear cell carcinomas. We found associations between c-myc copy-number changes and clinicopathological parameters of ovarian tumours such as degree of malignancy and histological type. We suggested that c-myc amplifications are characteristics for endometrioid, and c-myc gains for clear cell ovarian cancers. We suggest that copy-number increases of c-myc and 20q13.2 represent a possible mechanism for the regulation of the pathway STK15--c-myc--hTERT.

[First facioscapulohumeral muscular dystrophy prenatal diagnosis in a Bulgarian family].

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common myopathy. It is characterized by progressive descendent involvement of facial, shoulder girdle, truncal and lower extremities muscles. FSHD locus was mapped on the terminal part of the long arm of chromosome 4 (4q35). The disease is caused by a deletion of an integral number of tandem D4Z4 repeats and dimension of the pathological fragments < or = 38kb. Prenatal diagnosis of FSHD is possible but it is potentially difficult because of the big amount and high quality of DNA required. Hereby we describe the first prenatal tests performed for a Bulgarian family.

Association of 20q13.2 copy number changes with the advanced stage of ovarian cancer-tissue microarray analysis.

Overrepresentations in 20q have been reported in a number of ovarian cancers by comparative genomic hybridization. In order to study the relation of the increased copy number of 20q13.2 with tumor phenotype in ovarian cancer, we applied FISH on a tissue microarray. The TMA technology enables us to analyze a large number of different malignancy, histology, stage and grade tumors. Overall, the frequency of 20q13.2 alterations in epithelial ovarian cancer was 25.50% (10.74% gains and 14.76% amplifications). There was not statistically significant difference between the frequencies of 20q13.2 copy number changes in different grade tumors. The frequency of gains and amplifications increased significantly from stage I to stage II to stage III tumors. Our results showed strong association between increases 20q13.2 copies and advanced tumor stage. We concluded that genetic alterations in 20q13.2 may be of prognostic significance for stage progression of the ovarian cancer.

Tissue microarray analysis of cyclin D1 gene amplification and gain in colorectal carcinomas.

Colorectal cancer is one of the most common neoplastic diseases and one of the leading causes of cancer-related deaths. Elevated beta-catenin levels in colorectal cancer result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of the CCND1 gene. Overexpression of cyclin D1 is observed in one third of colorectal tumors. CCND1 amplification is the main cause of protein overexpression in numerous human carcinomas. In colorectal cancer, however, no CCND1 amplification has been reported so far. The aim of this study was to determine the frequency of CCND1 amplifications and gains in a large number of colorectal carcinomas, arranged in a tissue microarray, in order to assess their role in colorectal cancer development. The copy number changes, detected by fluorescence in situ hybridization, were predominantly gains (7.6%) and only rarely amplifications (2.5%). In colorectal cancer, the CCND1 copy number increase was neither associated with the tumor phenotype (stage and grade) nor with the tumor localization (colon, rectum or sigmoid colon). In conclusion, even in a small number of colorectal tumors, CCND1 gene amplification is a possible mechanism for the increase in cyclin D1 oncoprotein.

[Intranatal asphyxia, prematurity and congenital infection--their role for brain damage in very low birth weight newborns]. / Intranatalna asfiksiia, nedonosenost i vrodena infektsiia--roliata im za mozuchnite uvrezhdaniia pri detsa s mnogo nisko teglo pri razhdaneto.

The authors examined 273 newborns weighing less than 1500 g. Their birth weight was very low (1001-1500 g) in 179 but extremely low (< 1000 g) in 94 newborns. Neurological damage was proved in 101 cases (36.99%). The probable reasons for intra- (peri-) ventricular and cerebral hemorrhages in these newborns were the following: respiratory distress syndrome (in 11 cases or 10.89%), mother-fetal infection (in 16 cases or 15.84%), and combination of asphyxia and infection (in 10 cases or 9.9%). The severe degree of prematurity (and immaturity) remained the only causative factor in the rest 64 premature newborns (63.37% of the cases). The results from the distribution of the neurological lesions according to the gestational age were also considered. Usage of monofactorial regression models detected statistically significant differences between asphyxia, infection and brain damage in the newborns of different gestational age.

Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats.

In vivo cell interactions between Staphylococcus aureus and rat alveolar macrophages were investigated after intranasal inoculation during a 30-days period of examination. Some dynamic characteristics of microorganisms in the macrophages were examined by electron microscopy and acid phosphatase cytochemistry. It was found that at earlier infection intervals (days 3 and 7) the ingested cocci were sequestered in phagosomes and phagolysosomes and later many of the microbial cells were digested. An interesting finding was the intracellular appearance of cell wall-defective forms (L-forms) of S. aureus at later intervals (days 14 and 30 after challenge). Infection kinetics were evaluated by isolation and enumeration of colony-forming units of S. aureus from bronchoalveolar fluid and by assessment of blood and bronchoalveolar total and differential leukocyte counts. The results indicate that induction and survival of S. aureus L-forms may occur spontaneously in vivo. This phenomenon could explain some of the mechanisms, provoking the latent and relapsing lung infections.

Cell-wall-deficient forms of Staphylococcus aureus as lung pathogens: an ultrastructural study.

The interaction between stable protoplast L forms of Staphylococcus aureus and the alveolar wall of infected rats was observed in the course of experimental pulmonary infection (days 3, 7, 14, and 30 p.i.). The L forms were successfully cultivated from bronchoalveolar lavage samples taken throughout the tested interval. The ultrastructural results demonstrated the ability of the L forms to invade the alveolar wall where they established, grew and reproduced mainly in the interstitium. The infection caused lung lesions: granulomas, focal fibroses and destruction of type I alveolar epithelial cells.

Projected cancer incidence rates in Bulgaria, 1968-2017.

BACKGROUND: Incidence predictions are applicable when planning preventive or screening health care programmes, diagnostic, treatment and rehabilitation facilities. The aim of this study was to predict future (1993-2017) incidence rates of the most common sites of cancer in Bulgaria: breast, cervix and corpus uteri in females, and lung, prostate and stomach in males. METHOD: Observed numbers of incident cases in the period 1968-1992 and observed (predicted) population size were employed. Age-cohort and age-cohort-period log-linear models were fitted to the observed data, assuming no change in the observed trends. RESULTS: The incidence rates for all the studied primary sites, except stomach cancer, were predicted to increase. The observed rates in the period 1988-1992 and the predicted rates in the period 2013-2017 per 100000 were in females: breast-from 38.8 to 64.6, cervix-from 12.8 to 19.3, corpus uteri-from 12.4 to 26.5. In males similar rates were: lung-from 41.0 to 73.8, prostate-from 10.1 to 15.0 and stomach-from 17.5 to 10.2. Due to the increasing incidence rates and ageing of the population the predicted number of new cases in the studied sites of cancer in the period 2013-2017 is 62% higher than that observed in the period 1988-1992.