MiRNA Signatures Related to Invasiveness and Recurrence in Patients With Non-Functioning Pituitary Neuroendocrine Tumors.

PURPOSE: This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. METHODS: Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). RESULTS: Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. CONCLUSION: This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.

Altered Thermal Behavior of Blood Plasma Proteome Related to Inflammatory Cytokines in Early Pregnancy Loss.

Early pregnancy loss (EPL) is a relatively common pathology of which almost 50% of cases remain idiopathic. In the search for novel biomarkers, differential scanning calorimetry (DSC) is intensively used to characterize the thermodynamic behavior of blood plasma/serum proteome in health and disease. Herein, for the first time, we investigate the DSC denaturation profiles of blood plasma derived from patients suffering EPL compared to healthy pregnant and non-pregnant women. Data analysis reveals that 58% of the EPL thermograms differ significantly from those of healthy pregnant women. Thermal stabilization of a fraction of albumin-assigned transition with concomitant suppression of the major and enhancement of the globulin-assigned transition are characteristic features of EPL calorimetric profiles that could be used as a new indicator of a risk pregnancy. The presented results suggest an altered composition or intermolecular interactions of the plasma proteome of women with EPL. In addition, the alterations of the EPL thermograms correlate with the increased blood levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and a higher prevalence of the polymorphism in the plasminogen activator inhibitor type-1 (PAI-1) gene, suggesting an expression of an overall enhanced immune response. The concomitant changes in plasma thermograms confirm the potential of the DSC approach for distinguishing changes in the pathological state of the blood plasma proteome.

Identification of 99% of CFTR gene mutations in Bulgarian-, Bulgarian Turk-, and Roma cystic fibrosis patients.

BACKGROUND: The spectrum and frequencies of CFTR mutations causing Cystic fibrosis (CF) varies among different populations in Europe, and beyond. METHODS: We identified 98.9% of all CFTR mutations in a representative cohort of 140 CF patients comprising 107 Bulgarian- (BG), 17 BG Turk-, and 16 BG Roma cases. The compiled clinical and genotype dataset includes 110 previously analyzed patients with 30 cases currently analyzed for rare CFTR variants by massively parallel sequencing of the entire CFTR coding region and adjacent introns combined with the analysis of intra-CFTR rearrangements. RESULTS: Altogether 53 different mutations, of which 15 newly identified in the BG CF population, were observed. Comparison of clinical and laboratory data between individual BG ethnic groups proved that BG Roma have a more severe nutritional status and are younger than other CF patients, as well as that the spectrum mutations differs between them. CONCLUSION: This collaborative study improves genetic counselling in BG, facilitates introduction of multitier CF neonatal screening and fosters public health measures for improvement of care in the Roma CF population.

Genetically Determined TNF-α Secretion Influences the Development of Dermatomyositis and Systemic Lupus Erythematosus.

BACKGROUND: Abnormal secretion of TNF-α is known to play a role in the pathogenesis of dermatomyositis and systemic lupus erythematosus. MATERIALS AND METHODS: In the present study we have analyzed the concentrations of TNF-α in the sera of 30 patients with systemic lupus erythematosus (SLE), 28 with dermatomyositis (DM) and 30 healthy controls by standard ELISA tests. RESULTS: We have found that -308A allele increases TNF-α secretion, while -1031C and -863A alleles decrease it. The -857C/T and 489G/A polymorphisms appeared in strong linkage disequilibrium (D'=0.93) but they did not seem to affect TNF-α secretion. CONCLUSION: TNF-α polymorphisms play a significant role in its secretion and influence the development of DM and SLE.

Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson's Disease Pathogenesis.

Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B resulting in copper overload in the liver and brain. Direct sequencing is routinely used to confirm WD diagnosis; however, partial and whole gene deletions in the heterozygous state cannot be detected by exon amplification since the normal allele will mask its presence. The aim of the present work was to search for unusual mutational events in the unexplained WD cases and to provide insight into the mechanisms. Out of 1420 clinically and biochemically confirmed WD samples received between 2000 and 2014 for routine mutation analysis, we were unable to detect mutant alleles in 142 samples, after extensive sequencing analysis. We used selective amplification and MLPA to identify the partial gene deletions and identified three different partial gene deletions in seven different families. All three deletions were fully characterized at the DNA sequence level. We report the first hemizygous case with WD due to intragenic deletion in the ATP7B (c.3134_3556+689del). This novel deletion resulted from an excision event mediated by consensus sequences in an AluSq2 repeat element and could be traced to micro homologous end joining (MMEJ). Finally, we determined the prevalence of the three deletions in DNA samples from a multinational group of WD patients. Our results emphasize the need for searching mutant alleles beyond routine methods and highlight that large ATP7B deletions are rare, but account for a detectable proportion in some WD patients. Screening for gene aberrations will further improve mutation detection in patients with unidentified ATP7B mutations presenting with clinical manifestations of WD.

Association Between IL-6 and MMP3 Common Genetic Polymorphisms and Idiopathic Scoliosis in Bulgarian Patients: A Case-control Study.

STUDY DESIGN: A case-control study was performed on 105 patients with idiopathic scoliosis (IS) and 210 unrelated gender-matched controls from Bulgarian population. OBJECTIVE: Investigation of the association between common genetic polymorphisms of IL-6 and MMP3 genes and the etiology and progression of IS among Bulgarian patients. SUMMARY OF BACKGROUND DATA: The IL-6 and MMP3 genes have been considered as candidate genes of IS in Caucasian population. METHODS: Molecular detection of the promoter polymorphisms of IL-6 and MMP3 was performed by polymerase chain reaction followed by restriction fragment length polymorphism. The statistical analysis was performed by χ test with a value of P < 0.05 as statistically significant. The combinatorial effect of the candidate genes was also examined. RESULTS: This case-control study revealed statistically significant association between the IL-6 (rs1800795) functional polymorphism and susceptibility to IS (χ = 16.055; P < 0.0001). In addition, a significant association between IL-6 (rs1800795) and curve severity was detected (χ = 16.87; P < 0.0001). No genotype or allele of MMP3 (rs3025058) was found to be correlated to the onset or progression of IS (P > 0.05). One IL-6-MMP3 genotype combination was associated with the susceptibility to IS. CONCLUSION: IL-6 gene could be considered as a susceptibility and modifying factor of IS. The identification of molecular markers with diagnostic and prognostic value could be useful for early detection of children at risk for the development of IS and for prognosis of the risk for a rapid deformity progression. That would facilitate the therapy decisions and early stage treatment of the patient with the least invasive procedures. LEVEL OF EVIDENCE: 4.

Role of the IL-6 gene in the etiopathogenesis of idiopathic scoliosis.

Scoliotic human nuclei pulposi can respond to exogenous proinflammatory stimuli by secreting increased amounts of interleukin-6 (IL-6). The G/C polymorphism of the promoter region of IL-6 gene influences levels and functional activity of the IL-6 protein. We conducted a case-control study of eighty patients with idiopathic scoliosis (IS) and one hundred sixty healthy unrelated gender-matched controls trying to investigate the association between IS and the IL-6 promoter polymorphism at -174 position (rs1800795 G/C) in Bulgarian population. Molecular detection of the IL-6 genotypes was performed by amplification followed by restriction technology. The statistical analysis was performed by Pearson's chi-squared test. Our case-control study revealed a statistically significant association between the IL-6 (-174 G/C) functional polymorphism and susceptibility to IS. In addition, a significant association between the IL-6 (-174 G/C) polymorphism and curve severity was detected. IL-6 gene could be considered as susceptibility and modifying factor of idiopathic scoliosis. The identification of molecular markers with diagnostic and prognostic value could be useful for early detection of children at risk for the development of scoliosis and for prognosis of the risk for a rapid deformity progression. That would facilitate the therapy decisions and early stage treatment of the patient with the least invasive procedures.

Epidermal growth factor receptor mutations in East European non-small cell lung cancer patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations are recurrently observed in non-small cell lung carcinomas (NSCLCs), and it has been found that they may serve as specific therapeutic targets. The aim of the present study was to determine the prevalence of EGFR gene mutations in NSCLCs in an East European (Bulgarian) population in different histological subtypes, in cytological versus histological samples and in primary versus metastatic lesions. METHODS: In this study 1427 NSCLC samples were included. DNA was extracted from either formalin-fixed paraffin embedded (FFPE) tissues or cytology specimens and analyzed for the presence of 29 recurrent EGFR gene mutations using SARMS PCR. RESULTS: EGFR gene mutations were found to occur significantly more often in female than in male patients (19.4% vs. 5.4%; p<0.001), in adenocarcinomas than in squamous cell carcinomas or other histological subtypes (12.5% vs. 6.2%, and 7.6%, respectively; p=0.009), and in never smokers than in ex-smokers and current smokers (22.9% vs. 8.5% and 4.9%, respectively; p<0.001). No significant differences were observed in the occurrence of EGFR gene mutations in primary tumors compared to metastases (7.9% vs. 11.2%; p=0.092), or in FFPE samples compared to cytological samples (8.9% vs. 8.1%; p=0.813). CONCLUSIONS: Our data show that the overall frequency of EGFR gene mutations in lung adenocarcinomas in the East European cohort studied is within the range of that observed in North American and West European populations, but that its frequency in squamous cell carcinomas is higher than that in any population reported to date. All specimens appeared to be suitable for EGFR gene mutation analysis, irrespective nature or origin.

Association between Estrogen Receptor Alpha Gene Polymorphisms and Susceptibility to Idiopathic Scoliosis in Bulgarian Patients: A Case-Control Study.

BACKGROUND: The current consensus on idiopathic scoliosis maintains that it has a multifactorial etiology with genetic predisposing factors. AIM: Estrogen receptor alpha gene has been considered as candidate gene of idiopathic scoliosis. MATERIAL AND METHODS: We conducted a case-control study of Bulgarian population samples (eighty patients with idiopathic scoliosis and one hundred-sixty healthy unrelated gender-matched controls) trying to investigate the association between common genetic polymorphisms of estrogen receptor alpha and the susceptibility to idiopathic scoliosis. Molecular detection of the restriction polymorphisms XbaI and PvuII was performed by polymerase chain reaction following by restriction fragment length polymorphism. The statistical analysis was performed by Pearson's chi-squared test. RESULTS: Our case-control study showed statistically significant association between the PvuII polymorphism and susceptibility to idiopathic scoliosis and curve progression. No genotype or allele of XbaI polymorphism was found to be correlated with the onset or severity of the disease. CONCLUSIONS: The identification of molecular markers with diagnostic and prognostic value could be useful for early detection of children at risk for the development of scoliosis and for prognosis of the risk for a rapid deformity progression. That would facilitate the therapy decisions and early stage treatment of the patient with the least invasive procedures.

A Novel SRY Gene Mutation p.F109L in a 46,XY Female with Complete Gonadal Dysgenesis.

46,XY complete gonadal dysgenesis (CGD) is a disorder of sexual development that can result from different mutations in genes associated with sex determination. Patients are phenotypically females, and the disease is often diagnosed in late adolescence because of delayed puberty. Here, we present the clinical and molecular data of a 46,XY female CGD patient with gonadoblastoma with dysgerminoma and incidentally found inherited thrombophilia. The clinical significance of the described de novo SRY gene mutation c.325T>C (p.F109L) is discussed. This case report supports the critical role of the HGM domain in the SRY gene and the need of a multidisciplinary approach for CGD patients.

Rare mutations in the PIK3CA gene contribute to aggressive endometrial cancer.

The molecular basis of endometrial cancer (EC), a common gynecologic malignancy, often includes mutational activation of the PIK3CA and KRAS genes. We aimed to determine the distribution of mutations in the two genes depending on patient clinocopathological characteristics. We sequenced exon 1 of the KRAS gene and exons 9 and 20 of the PIK3CA gene in 108 consecutive EC tumor samples. PIK3CA mutations were present in 24 of the 108 (22.2%) cases and KRAS mutations in 18 of the 108 (16.7%) cases. PIK3CA mutations occurred more frequently in KRAS-mutated samples (7/18, 38.9%; p = 0.06) than in KRAS wild type (17/90, 18.9%) and showed a very high frequency in metastatic tumors (4/9, 44.4%; p = 0.1) and in samples displaying serous differentiation-serous and mixed endometrioid/serous tumors (6/12, 50.0%; p = 0.021)-where KRAS mutations were rare (11.1% and 16.7%, respectively) and did not exist independently of a PIK3CA mutation. Non-hotspot (i.e., non-E542K, -E545K, and -H1047R) mutations in the PIK3CA gene showed higher frequency in metastatic cases (3/9, 33.3%; p = 0.05). Tumors displaying serous differentiation showed a particular pattern-they harbored exclusively mutations in PIK3CA exon 20 (5/12, 41.7%; p = 0.005) and most of these were non-hotspot (4/12, 33.3%; p = 0.02). In all other comparisons exons 9 and 20 mutation distribution did not differ. These results suggest the need for further exploration of the significance of PIK3CA mutations in respect to aggressive EC.

Vitamin D and estrogen receptor gene polymorphisms and the risk of colorectal cancer in Bulgaria.

BACKGROUND AND AIMS: Different epidemiological studies report the protective effect on colorectal cancer (CRC) exerted by vitamin D(3) intake, estrogen replacement therapy, and increase of the risk of microsatellite instability (MSI) in CRC by withdrawal of estrogens. The aim of our study was to search for association between CRC and polymorphisms in estrogen receptor-alpha (ER-alpha) and Vitamin D receptor (VDR) genes. MATERIALS AND METHODS: We analyzed the PvuII and XbaI polymorphisms from the ER-alpha gene and the BsmI polymorphism of the VDR gene in 140 patients with CRC (subsequently divided according to their MSI status) and 94 controls. RESULTS: We have demonstrated that the presence of the PvuII pp genotype increased the risk of developing MSI(+) tumors about three times compared to MSI(-) tumors [odds ratio (OR)=3.09, 95% confidence interval (CI) 0.88-10.91]. The effect of the XbaI xx genotype was similar (OR=2.08, 95% CI 0.49-8.81). Our results for the VDR BsmI polymorphism showed an increased risk for CRC in bb carriers (OR=1.8, 95% CI 0.81-4.05). CONCLUSION: We conclude that PvuII and XbaI polymorphisms in the ER-alpha gene were associated with the risk of developing MSI in CRC patients. The BsmI polymorphism in the VDR gene was linked to the risk of developing CRC.