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Kadiyska T.K.,Medical University-Sofia | Kadiyska T.K.,Genetic Medico Diagnostic Laboratory Genica | Todorov T.P.,Genetic Medico Diagnostic Laboratory Genica | Bichev S.N.,Sofia University | And 4 more authors.
Clinical Genetics | Year: 2014

Over 1500 adenomatous polyposis coli (APC) gene mutations have already been identified as causative of familial adenomatous polyposis (FAP). However, routine genetic testing fails to detect mutations in about 10% of classic FAP cases. Recently, it has been shown that a proportion of mutation-negative FAP cases bear molecular changes in deep intronic and regulatory sequences. In this study, we used direct sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) of genomic DNA from family members, affected by classic FAP. We first reported the family as mutation negative. With the launch of a new version of MLPA kit, we retested the family and a novel full deletion of promoter 1B was detected. The exact breakpoints of the deletion were determined by array comparative genomic hybridization (CGH) and long range polymerase chain reaction (PCR), followed by direct sequencing. The total APC expression levels were investigated by quantitative polymerase chain reaction (qPCR) assay and allele-specific expression (ASE) analysis. The APC gene expression was highly reduced, which indicates causative relationship. We suggest that there is a significant possibility that APC promoter 1B mutations could be found in mutation-negative FAP patients. In the light of our findings it seems reasonable to consider targeted genetic re-analysis of APC promoter 1B region in a larger cohort of unsolved cases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.


Lukova M.,Medical University-Sofia | Lukova M.,Genetic Medico Diagnostic Laboratory Genica | Todorova A.,Medical University-Sofia | Todorova A.,Genetic Medico Diagnostic Laboratory Genica | And 2 more authors.
Molecular Biology Reports | Year: 2013

Molecular abnormalities in the 11p15.5 imprinted gene cluster lead to two different growth diseases: Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). They are mainly caused by epigenetic alterations in one of the two imprinting 11p15 control regions (ICR1 and ICR2). These CpG-rich regions are differentially methylated on the maternally and paternally derived chromosomes. We report four different methylation patterns along the BWS/SRS critical region, clarified by methylation-specific multiplex ligation-dependent probe amplification. The mathematical processing of the data provides information about alterations in the methylation status: from hypo- to almost complete demethylation of KvDMR, hypo- and hypermethylation of H19DMR and combined results from both regions provide information on paternal uniparental disomy (patUPD). The study concerns two BWS cases with KvDMR hypomethylation and almost complete loss of methylation, respectively; two patUPD11p15 cases with H19DMR hypermethylation/KvDMR hypomethylation, and one SRS case with H19DMR demethylation. In some cases KvDMR hypomethylation in patUPD11p15 can be difficult to assess, which requires combination with STR analysis or alternative method. The STR analysis provides also information on complete or segmental coverage and iso- or heterodisomy. Following this systematic approach, the precise diagnosis can be clarified in a few days and different methylation patterns could be detected. © 2012 Springer Science+Business Media Dordrecht.


Karayasheva D.,Medical University-Sofia | Glushkova M.,Genetic Medico Diagnostic Laboratory Genica | Boteva E.,Medical University-Sofia | Mitev V.,Medical University-Sofia | And 2 more authors.
Archives of Oral Biology | Year: 2016

Objective Various exogenous and endogenous risk factors have been described as contributing to dental caries susceptibility. In the last decade it has been established that both pro and active forms of host derived Matrix metalloproteinases (MMPs) are present in the oral cavity. MMPs role in caries development has been hypothesized. The aim of this study was to analyse MMP2 (rs2287074) and MMP3 (rs679620) single nucleotide polymorphisms (SNPs) and their role in caries susceptibility. Design The two SNPs were analysed by PCR- restriction fragment length polymorphism (RFLP) in a sample of 102 ethnic Bulgarian volunteers (42 males and 60 females), all students in Sofia Medical University. Results Statistical analysis of the MMP2 SNP showed significant differences for the genotype frequencies between the caries free (CF, DMFT = 0) and low caries experience (LCE, DMFT ≤ 5) groups. Analysis for the non-synonymous MMP3 SNP found significant differences between both CF vs caries experience groups (LCE+ high caries experience (HCE, DMFT ≥ 5)) and LCE vs HCE groups. The presence of allele G decreased the risk of HCE about 4 times. Conclusions MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is a multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome. © 2016 Elsevier Ltd. All rights reserved.


Dimova P.S.,Sofia University | Kirov A.,Medical University-Sofia | Kirov A.,Genetic Medico Diagnostic Laboratory Genica | Todorova A.,Medical University-Sofia | And 4 more authors.
Pediatric Neurology | Year: 2012

We report on a 13-year-old girl with a negative family history who manifested drug-resistant, mostly fever-induced seizures in clusters from age 5 months. Seizure frequency was not substantially reduced by anticonvulsant treatment, but tended to decrease with age. Early behavioral changes, i.e., autistic and aggressive features, worsened with time. Molecular genetic testing for PCDH19 mutations was performed by sequencing all exons of the gene, and revealed duplication c.2705dupA (p.Asp902Lysfs*6) in exon 5, which was also present in the fully asymptomatic mother. This case is among the few reported with a pathogenic PCDH19 mutation inherited from an unaffected heterozygous female carrier. It indicates that PCDH19 mutation testing should be performed in sporadic cases with no family history that still demonstrate well-established features of peculiar X-linked epilepsy with mental retardation limited to females. © 2012 Elsevier Inc. All rights reserved.


Kirov A.,Medical University-Sofia | Kirov A.,Genetic Medico Diagnostic Laboratory Genica | Dimova P.,Sofia University | Todorova A.,Medical University-Sofia | And 8 more authors.
Epilepsy Research | Year: 2013

Purpose: The chromosome 15q13.3 region is a genomic rearrangement hotspot linked to idiopathic generalized epilepsies (IGEs) and such rearrangements remain the strongest risk factor for IGE known to date. Increasing evidence suggests that genetic variations can be highly population-specific. Therefore, we aimed to assess the frequency of 15q13.3 microdeletions in IGE patients from Bulgaria. Methods: A cohort of 100 patients with various IGE syndromes was screened for large deletions/duplications by MLPA. All deletions and duplications were confirmed by array CGH analysis as previously described. Results: In 100 prospectively recruited Bulgarian patients with IGE, we found one case with a microdeletion, which amounted to 1% frequency for this copy number variant. Conclusion: We confirm the frequency of 1% for the 15q13.3 microdeletion in a prospectively recruited cohort of Bulgarian epilepsy patients, demonstrating that this variation represents a significant risk factor for IGE for various populations and that it is retrospectively detected frequency is not due to selection bias. © 2013 Elsevier B.V.


PubMed | Medical University-Sofia and Genetic Medico Diagnostic Laboratory Genica
Type: | Journal: The Journal of pediatrics | Year: 2016

Ten girls with sporadic central precocious puberty were screened for mutations in the maternally imprinted gene MKRN3. We detected 1 novel frameshift mutation (p.Arg351Serfs*44) and a previously described mutation (p.Pro161Argfs*10). In the course of investigating the family, genetic analysis found 2 asymptomatic males with paternally inherited MKRN3 mutations, which has not been reported in previous studies.


PubMed | Medical University-Sofia and Genetic Medico Diagnostic Laboratory Genica
Type: | Journal: Archives of oral biology | Year: 2016

Various exogenous and endogenous risk factors have been described as contributing to dental caries susceptibility. In the last decade it has been established that both pro and active forms of host derived Matrix metalloproteinases (MMPs) are present in the oral cavity. MMPs role in caries development has been hypothesized. The aim of this study was to analyse MMP2 (rs2287074) and MMP3 (rs679620) single nucleotide polymorphisms (SNPs) and their role in caries susceptibility.The two SNPs were analysed by PCR- restriction fragment length polymorphism (RFLP) in a sample of 102 ethnic Bulgarian volunteers (42 males and 60 females), all students in Sofia Medical University.Statistical analysis of the MMP2 SNP showed significant differences for the genotype frequencies between the caries free (CF, DMFT=0) and low caries experience (LCE, DMFT5) groups. Analysis for the non-synonymous MMP3 SNP found significant differences between both CF vs caries experience groups (LCE+ high caries experience (HCE, DMFT5)) and LCE vs HCE groups. The presence of allele G decreased the risk of HCE about 4 times.MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is a multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome.


PubMed | Medical University-Sofia, Sofia University and Genetic Medico Diagnostic Laboratory Genica
Type: Journal Article | Journal: Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology | Year: 2015

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by intractable seizures in neonates and infants. The seizures cannot be controlled with antiepileptic medications but respond both clinically and electrographically to large daily supplements of pyridoxine (vitamin B6). PDE is caused by mutations in the ALDH7A1 gene. Molecular genetic analysis of the ALDH7A1 gene was performed in seven patients, referred with clinical diagnosis of PDE. Mutations were detected in a dizygotic twin pair and a non-related boy with classical form of PDE. Direct sequencing of the ALDH7A1 gene revealed one novel (c.297delG, p.Trp99*) and two already reported (c.328C>T, p.Arg110*; c.584A>G, p.Asn195Ser) mutations. Here, we report the first genetically proven cases of PDE in Bulgaria.


PubMed | Medical University-Sofia and Genetic Medico Diagnostic Laboratory Genica
Type: Case Reports | Journal: Journal of child neurology | Year: 2015

A family with 2 siblings with severe spinal muscular atrophy with respiratory distress 1 (SMARD1) was genetically proved to be caused by mutations in IGHMBP2 gene. Both patients developed progressive muscular weakness and respiratory distress and died before 6 months of age. One novel deletion, c.780delG;p.(Gln260Hisfs*24), inherited from the father and a nonsense mutation, c.1488C>A;p.(Cys496*), inherited from the mother were detected. An attempt was made to correlate the genetic-clinical data available in the literature. The clinical case presented in this study might be considered as the most severe form of spinal muscular atrophy respiratory distress 1 reported so far, presumably because of the total absence of IGHMBP2 enzyme activity.


PubMed | Genetic Medico Diagnostic Laboratory Genica
Type: Editorial | Journal: World journal of gastroenterology | Year: 2015

Colorectal cancer (CRC) is fourth most common cancer in men and third in women worldwide. Developing a diagnostic panel of sensitive and specific biomarkers for the early detection of CRC is recognised as to be crucial for early initial diagnosis, which in turn leads to better long term survival. Most of the research on novel potential CRC biomarkers in the last 2 decades has been focussed on stool DNA analysis. In this paper, we describe the recent advances in non-invasive CRC screening and more specifically in molecular assays for aberrantly methylated BMP3 and NDRG4 promoter regions. In several research papers these markers showed superior rates for sensitivity and specificity in comparison to previously described assays. These tests detected the majority of adenomas 1 cm in size and the detection rates progressively increased with larger adenomas. The methylation status of the BMP3 and NDRG4 promoters demonstrated effective detection of neoplasms at all sites throughout the colon and was not affected by common clinical variables. Recently, a multitarget stool DNA test consisting of molecular assays for aberrantly methylated BMP3 and NDRG4 promoter regions, mutant KRAS and immunochemical assay for human haemoglobin has been made commercially available and is currently reimbursed in the United States. Although this is the most sensitive non-invasive CRC screening test, there is the need for further research in several areas - establishment of the best timeframe for repeated DNA stool testing; validation of the results in populations outside of North America; usefulness for surveillance and prognosis of patients; cost-effectiveness of DNA stool testing in real-life populations.

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