Introduction: Cardiomyopathies are a clinically and genetically heterogeneous group of diseases, that are associated with significant morbidity and mortality. The aim of the present study is to clarify the molecular-genetic characteristics of cardiomyopathies in patients in Bulgaria. Material and methods: In the present study, targeted analysis of an expanded panel of 242 genes, associated with cardiomyopathy, and an additional panel of 20 genes, associated with hereditary amyloidosis, was performed in a total of 20 Bulgarian patients, diagnosed with cardiomyopathy, as follows: 12 patients with hypertrophic cardiomyopathy (HCM), including 1 pediatric patient, 6 patients with dilated cardiomyopathy (DCM), of whom 2 pediatric patients, and 2 patients with restrictive cardiomyopathy (RCM). Family segregation analyses were performed by direct Sanger sequencing. Results: Genetic findings were present in 90% of the patients. Pathogenic/likely pathogenic variants were found in 12 of the patients (60%), while genetic findings related to the clinical symptoms were not detected in the RCM patients (10%). Approximately 1/3 of the patients had a family history of sudden cardiac death or cardiomyopathy. Pathogenic/likely pathogenic variants were found in 55% of HCM patients with no family history, in ~67% of HCM patients with family history or with sporadic DCM, and in 100% of DCM patients with a positive family history. A respectively 2,5 and 4-fold higher frequency of truncating variants was found in the study group compared to the reports of around 10% in the literature, both in patients with HCM, and in patients with DCM. Pathogenic/likely pathogenic variants in the MYBPC3 gene (71%) were found with the highest frequency in HCM, while DCM is characterized by a diverse genetic profile, and genetic findings in the NDUFB11 and TAZ genes were associated with severe clinical presentation in pediatric patients in the first postnatal days. Results of segregation analyses were reported in 6 of the affected families. Conclusion: The data from the present study supports the importance of conducted genetic testing and medical-genetic counseling in patients and affected families with cardiomyopathy in Bulgaria

We present the findings of a Whole Exome Sequencing in a 2-year-old boy, conceived via In Vitro Fertilization with donor sperm, who suffers from an undiagnosed neurological syndrome. The following heterozygous variant in the EPHA4 gene was identified and classified as likely pathogenic: c.1655_1656, p.(Ser552CysfsTer23). Subsequent segregation analysis showed that the variant was not inherited from the mother and the sperm donor is not accessible for genetic testing. The presented results can further expand upon the genetic variants considered when diagnosing complex neurological syndromes and shows the importance of access to biological samples from donor banks in genetically ambiguous cases.

To investigate the potential role of the following bacterial/viral panel (Chlamydia trachomatis, Ureaplasma urealyticum/parvum, Mycomplasma hominis/genitalium, Gardnerella vaginalis, HSV1/2, EBV, CMV, VZV, HHV6, HHV7, HHV8) as causative factors for miscarriages in women by testing endometrial biopsies. Anaerobic and aerobic microorganisms causing dysbiosis and endometrial bacterial colonization by unbalanced growth were additionally tested.

Abstract Aim: We investigated the role of bacterial (Chlamydia trachomatis, Ureaplasma urealyticum/ parvum, Mycoplasma hominis/genitalium, Gardnerella vaginalis) and viral (HSV1/2, EBV, CMV, VZV, HHV6/HHV7, HHV8) pathogens, as a potential cause of reproductive failure in women by testing menstrual tissue samples. Materials and methods: We examined DNA extracted from 180 probands selected on the basis of their infertility background. DNA extraction, real-time qPCR, agarose gel-electrophoresis were applied. Results: In 61,1% of all tested menstrual tissue samples of infertile women bacterial and/ or viral pathogens were detected. In 48,8% of all tested samples we found bacterial, while in 22,2% viral pathogens. Ureaplasma parvum and Gardnerella vaginalis were detected in 61,36% and 69,31%, respectively, while Mycoplasma hominis and Ureaplasma urealyticum in equivalent rate 2,27% of the positive for bacterial pathogens samples. HHV7, EBV, HHV6, CMV, HSV2 were detected in 42,5%, 40%, 10%, 7,5%, 2,5% of the positive for viral factors samples. Variable combinations of bacterial and/or viral co-infection were found in 39,9% of all infected patients. Infections with the rest of target pathogens were not detected in the menstrual tissue samples. Conclusions: Our research offers new approach for noninvasive diagnostics of infections in the upper female genital tract by analyzing menstrual tissue as a target biological sample. The detection of asymptomatic bacterial and viral infections or co-infections in female endometrium contributes to the clarification of the infectious etiology of reproductive failure. It is of a great importance for applying of an adequate and individualized therapy.

Abstract Objective The aim of this study was to investigate the potential association of rs11206510 in PCSK9 gene with coronary artery disease (CAD) and myocardial infarction (MI) in Bulgarians. Materials and Methods The current analysis included 261 patients with angiographically documented CAD (153 with MI and 108 without MI) and 496 population – based controls. Genomic DNA was extracted from venous blood samples. The selected polymorphism was genotyped by TaqMan SNP Genotyping Assay. The genotype and allele frequencies were compared between cases and controls using χ2 test. Results In this study, the presence of the T allele of rs11206510 in the PCSK9 gene was found to be associated with elevated risk for MI in patients with already existing myocardial ischemia (allele T, OR1.78,CI95:1.16-2.73, p = 0.007). The result was enhanced in the male subgroup (allele T, OR1.74, CI95:1.02-2.96, p = 0.038). Also, we found reduced risk of CAD (without MI) for T allele (OR0.70, CI95:0.49-0.99, p = 0.04). This trend was stronger in the male subgroup (OR0.56, CI95:0.35-0.90, p = 0.02). There was not any relationship of the studied genetic variant with the levels of total cholesterol, triglycerides, low density lipoproteins and high-density lipoproteins, or with systolic and diastolic blood pressure values. Conclusion Our study found a difference in the frequencies of rs11206510 genotypes and alleles in the PCSK9 gene between cases and controls, and the relationship of the investigated polymorphism to the risk of cardiac injury in the Bulgarian population was demonstrated. Further investigations with a larger number of cases and controls will be needed in order to evaluate a possible association between this variant and CAD/MI in Bulgarians.

NARS1-related neurodevelopmental disorder is caused by pathogenic variants in the NARS1 gene. The aim of the present study was to determine the genetic reason for the epilepsy and microcephaly, observed in the tested patient. Using whole exome sequencing two novel heterozygous variants c.676G>C, p.Val226Leu and c.986G>A, p.Arg329Gln in the NARS1 gene were detected. The obtained results from the molecular-genetic analysis can enrich the spectrum of epilepsy related variants and can explain the genetic etiology of a severe epileptic disease.

Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, characterized by progressive degeneration of the upper and lower motor neurons, leading to muscle weakness, hypotrophy, swallowing and respiratory failure. The cause of ALS is not yet fully elucidated, but there are 35 associated genes and 2 gene loci with an unidentified gene. The most common are C9orf72, SOD1, TARDBP and FUS found in approximately 10% of patients. Variants in the C9orf72 gene are the main cause of fALS – 25-40% of cases (and a small percentage of sALS). The goal of the present study was to evaluate the significance of the C9orf72 hexanucleotide repeat expansion in Bulgarian patients with ALS, through the means of in house and triplet repeat-primed PCR assay (TP-PCR). From 171 patients diagnosed with ALS and included in the current study, we have identified the repeat expansion with more than 145 GGGGCC repeats in 7 (4,1%). Short expansions or borderline values (24 to 30 repeats) were not detected. Due to absence of sufficient data, we have established an ALS-focused research for the association of the C9orf72 gene in clinically well-characterized Bulgarian ALS patients. Published data show variable percentage ratios for genetically verified cases (4-40%), which is mainly due to small sample counts and sALS-fALS ratios. Our patients’ group also contains sALS and fALS cases, which explains the low percentage of genetic verification. The obtained results enrich the worldwide database and shed light onto genetically characterized Bulgarian ALS patients. Affected patients and their families can receive adequate medical-genetic consultation and prenatal diagnostic testing.

Abstract Introduction: Thyroid carcinoma is the most common endocrine cancer. Some somatic mutations in genes (BRAF, NRAS and TERT) involved in key signaling pathways and genome stability have been recently identified to play an important role in its development. Very little research has been done on their frequency and clinical relevance in Bulgarian patients with papillary thyroid cancer (PTC). This study is focused on investigating somatic mutation frequency in Bulgarian patients with PTC and their association with clinicopathologic features. Material and Methods: The study included 50 PTC from Bulgarian patients analyzed for mutations in BRAF (V600E), NRAS (Q61K), single nucleotide polymorphism (SNP) rs2853669 and TERT (C228T and C250T) genes by Sanger sequencing. The results were interpreted using Benchling and SeqScape software, and statistical analysis performed with SPSS. Results: In the studied PTC group BRAF(V600E) and TERT (C228T) mutations were found with frequency of 24% and 2%, respectively. Co-occurrence of both mutations was found in 1 patient (2%). The mutations Q61K (NRAS), and C250T (TERT) were not detected. The SNP rs2853669 was found in 18 patients (52.9%). Correlation analysis with the clinical characteristics of the patients revealed statistically significant association with larger size of the tumor for BRAF(V600E) and smaller tumor size for rs2853669. Conclusion: In the present pilot study, we found that BRAF(V600E) and rs2853669 in TERT are common among PCT patients. While the presence of BRAF V600E mutation was associated with large tumors, the presence of rs2853669 in TERT was found in the majority of PCT below 2 cm. More extensive molecular genetic analysis of TERT, BRAF or RAS mutations in larger sample is needed to further elucidate the clinically important diagnostic and prognostic biomarkers for thyroid cancer.

Abstract Objective The aim of the study was to determine the molecular-genetic characteristics of the autosomal dominant systematic disorder Tuberous Sclerosis Complex (TSC1 and TSC2) in Bulgarian patients and to derive some genotype-phenotype correlations. Material and Methods In total 42 patients/families with suspected clinical diagnosis of TSC were analyzed. We used direct sequencing and MLPA for the TSC1 and TSC2 gene analysis. Results In 38 families (90.5%) we confirmed the suspected clinical diagnosis – 15 with TSC1 (35.7%) and 23 (54.8%) with TSC2. In 4 families (9.5%) pathogenic variants were not found. In all 38 patients with proven diagnosis of TSC, we found 38 different mutations, 15 of which (39%) were detected for the first time by our research group. The mutation “hotspots“ in TSC1 gene are exons 9, 15, 17 and 18, where 73% of the TSC1 mutations are localized, while the TSC2 gene mutation “hotspots“ are exons 13 and 34, with 22% of the mutations situated there. In the TSC2 patients the common clinical findings include subcortical tubers, epilepsy with generalized tonic-clonic seizures, subependymal giant cell astrocytoma, facial angiofibromas, ungual fibromas, cardiac rhabdomyomas and renal angiomyolipomas, while in the TSC1 patients typically cortical tubers, cortical dysplasia and subependymal nodules were registered. In patients with aggressive frameshift and nonsense TSC1 and TSC2 mutations commonly hypomelanotic macules, cortical and subcortical tubers, cortical dysplasia, epilepsy with different types of seizures were found. Renal angiomyolipomas and cysts were detected mainly in patients with large deletions. Shagreen patches and intellectual disability were typically registered in equal degree in patients with frameshift, nonsense and missense mutations. Conclusion Although some genotype-phenotype correlations were derived, there is a great inter- and intrafamilial clinical variability in TSC, so it is impossible to predict the course of the disease on the basis of the detected molecular defect. The obtained results helped us to develop a diagnostic algorithm for proper molecular-genetic diagnostics which permits adequate genetic counseling, prophylaxis and treatment in the affected TSC families.

Случаите на ранна малкомозъчна атаксия в резултат на дефицит на коензим Q (CoQ) се изявяват най-често в ранна детска възраст, като освен церебеларна атаксия и атрофия са възможни периферна невропатия, епилептични пристъпи, умствена изостаналост, дистония и миопатия. По-редки прояви са птоза и офталмоплегия, retinitis pigmentosa, сензоневрална загуба на слуха и ендокринологични нарушения. В статията е представен случай на 12-годишно момиче с доказана мутация в COQ8A с водеща клинична картина на церебеларна атаксия. Детето е родено от нормално протекли бременност и раждане, но е с изоставащо двигателно и невро-психично развитие – проходило е след навършване на 1 година и 8 месеца и е с изоставащо говорно развитие. По този повод на 4-годишна възраст е проведена компютърна томография (КТ), в която е налице церебеларна атрофия с широк четвърти вентрикул. На 8-годишна възраст при пациентката се изявява пристъп с характеристиките на генерализиран тонично-клоничен, във връзка с което е проведено лечение с валпроат. Състоя нието е проследявано с провеждане на нови КТ на главен мозък, в които се визуализира прогресия на церебеларната атрофия. При последната хоспитализация в Клиниката по нервни болести за деца, УМБАЛНП „Св. Наум”, когато момичето е на 12 години, неврологичният статус показва церебеларен синдром с интенционен тремор, дизметрия и атактична походка. Проведено е психологично изследване, което показва лека степен на умствена изостаналост при пациентката. Електромиографията и електроневрографията са без данни за периферна невропатия или миопатия. Генетичното изследване доказва мутация в екзон 7 на ген COQ8A. Своевременното доказване на дефицит на CoQ при пациенти с церебеларна атаксия е от особена важност поради възможното подобрение на симптоматиката при заместително лечение.

Here we report the first familial case spread through at least three generations, genetically verified cases of Marshall-Stickler syndrome in Bulgaria. The proband, a 2-year-old girl, has craniofacial dysplasia, ocular hypertelorism, small saddle nose with a flat bridge and midface hypoplasia. The pedigree of the proband’s family showed that her father has the same clinical manifestations of the disease. In addition, her father presented with a tall, thin stature and mild hearing loss, manifested with aging. The same dysmorphological symptoms were presented by the paternal grandfather. Both patients, the 2-year-old girl and her father, have been diagnosed to carry Marshall-Stickler syndrome. The COL2A1 gene tested negative in the family. Based on the higher percentage of mutations in the COL2A1 gene, we analyzed this gene as the first target in the family. The COL2A1 gene tested negative, and we sequenced the gene further. A novel splice site mutation c.3474+1G>A was found in intron 44. This variant is related to the clinical presentation in the patient and her father. The c.3474+1G>A mutation results in altered splicing affects at the donor splice site of intron 44, which most probably gives a nonfunctional protein. The variant affects the major triple-helical domain that represents a mutation hot-spot for the gene.

The process of neoangiogenesis is one of the classic hallmarks of a cancer. Its intricate mechanisms have long been one of the major domains in cancer research and a hope for a therapeutic breakthrough. Last decade a new subgroup of non-coding RNA molecules was reported called microRNAs. Literally hundreds of new molecules in this class are being uncovered as pivotal regulators in virtually all intracellular processes. The aim of this study is to classify and review those microRNA molecules that have a role in the processes of tumor angiogenesis and map their places in the regulatory framework of the classical proangiogenic genes and their canonical cascades.

Abstract Objective: The aim of the study was to determine the molecular mechanisms of mutagenesis in Bulgarian patients with Angelman syndrome (AS). AS is a severe neurodevelopmental disorder caused by loss of expression in brain of the maternally inherited UBE3A gene as a result of various 15q11.2-q13 alterations. Material and Methods: In total 24 patients (11 boys, 13 girls) from 22 unrelated families with suspected clinical diagnosis AS were analysed. We used methylation specific PCR, multiplex ligation-dependent probe amplification, methylation sensitive MLPA, and direct sequencing of the UBE3A gene. Results: In 9 families (41%) pathogenic mutations were detected, which confirmed the clinical diagnosis on а molecular-genetic level. In 4 (44%) of these families we found 15q11-q13 region deletion with breakpoints BP1-BP3 or BP2-BP3. In 1 (11%) of the families we found imprinting defect: deletion of the AS-SRO regulatory region (part of the PWS-AS imprinting center). In 1 (11%) of the families we detected a rare finding – paternal uniparental disomy of chromosome 15. In 3 (33%) of the families diff erent point mutations in the UBE3A gene were detected: two novel missence mutations c.488T > C; p.Leu163Ser and c.1832A > T; p.Gln611Leu, and one known frameshift mutation c.2576_2579delAAGA; p.Lys859Argfs*4. Conclusion: The obtained results helped us to develop a systematic diagnostic algorithm in order to provide proper diagnosis for the patients with AS. Combining excellent knowledge of the molecular mechanisms of mutagenesis and proper molecular-genetic testing approaches is a cornerstone in the management of AS patients, ensuring AS families would receive both adequate genetic counseling and prophylaxis of the disease in the future.