MLPA in the initial genetic screening of patients with acute myeloid leukemia.

INTRODUCTION: This study aimed to assess the effectiveness of multiplex ligase-dependent probe amplification (MLPA) in the initial genetic screening of patients with acute myeloid leukemia (AML) since current risk stratification and clinical management depend on molecular-genetic markers. METHODS: We performed a prospective case-control study on newly diagnosed patients from the Clinical hematology clinic of UMHAT "St. Marina", Varna, for the period 02.2022 - 02.2023. MLPA - a semiquantitative PCR-based method, was implemented with probes for 40 AML/myelodysplastic syndrome-typical genetic changes. We compared these findings with a parallelly carried out cytogenetic analysis, part of the routine diagnostic process. RESULTS: We assessed 61 patients - 29 females and 32 males, median age of 61 years for females and 65 for males (min-max 20-89). 34 (56%) of all showed pathological results, while the rest 27 (44%) did not. Of the 34, 22 (65%) had a single gene variant in genes NPM1, DNMT3A, FLT3, and IDH2, isolated or in combination. 18 (53%) of the same 34 also had copy number aberration (CNA) in chromosomes 4, 5, 6, 7, 11, 14, 17, and 21. The latter were either isolated or in combination with other findings. 8 of the 18 cases also underwent cytogenetic analysis, with concordance between the two methods in 4. CONCLUSION: MLPA is an informative method for initial genetic assessment in addition to cytogenetic analysis. Still, more patients are needed to draw finite conclusions on its eligibility for routine use. Given the significant percentage of normal results - 44%, simultaneous evaluation of more genetic markers, included in current guidelines, is reasonable.

Genus-Level Analysis of Gut Microbiota in Children with Autism Spectrum Disorder: A Mini Review.

Autism is a global health problem, probably due to a combination of genetic and environmental factors. There is emerging data that the gut microbiome of autistic children differs from the one of typically developing children and it is important to know which bacterial genera may be related to autism. We searched different databases using specific keywords and inclusion criteria and identified the top ten bacterial genera from the selected articles that were significantly different between the studied patients and control subjects studied. A total of 34 studies that met the inclusion criteria were identified. The genera Bacteroides, Bifidobacterium, Clostridium, Coprococcus, Faecalibacterium, Lachnospira, Prevotella, Ruminococcus, Streptococcus, and Blautia exhibited the most substantial data indicating that their fluctuations in the gastrointestinal tract could be linked to the etiology of autism. It is probable that autism symptoms are influenced by both increased levels of harmful bacteria and decreased levels of beneficial bacteria. Interestingly, these genera demonstrated varying patterns of increased or decreased levels across different articles. To validate and eliminate the sources of this fluctuation, further research is needed. Consequently, future investigations on the causes of autism should prioritize the examination of the bacterial genera discussed in this publication.

Oxford Nanopore Technology and its Application in Liquid Biopsies.

Advanced medical technologies are transforming the future of healthcare, in particular, the screening and detection of molecular-genetic changes in patients suspected of having a neoplasm. They are based on the assumption that neoplasms release small amounts  of  various  neoplasm-specific molecules, such as tumor DNA, called circulating DNA (cirDNA), into the extracellular space and subsequently into the blood. The detection of tumor-specific molecules and specific molecular changes in body fluids in a noninvasive or minimally invasive approach is known as "liquid biopsy." The aim of this review is to summarize the current knowledge of the application of ONT for analyzing circulating DNA in the field of liquid biopsies among cancer patients. Databases were searched using the keywords "nanopore" and "liquid biopsy" and by applying strict inclusion criteria. This technique can be used for the detection of neoplastic disease, including metastases, guiding precision therapy, and monitoring its effects. There are many challenges, however, for the successful implementation of this technology into the clinical practice. The first one is the low amount of tumor-specific molecules in the body fluids. Secondly, a tumor molecular signature should be discriminated from benign conditions like clonal hematopoiesis of unknown significance. Oxford Nanopore Technology (ONT) is a third-generation sequencing technology that seems particularly promising to complete these tasks. It offers rapid sequencing thanks to its ability to detect changes in the density of the electric current passing through nanopores. Even though ONT still needs validation technology, it is a promising approach for early diagnosis, therapy guidance, and monitoring of different neoplasms based on analyzing the cirDNA.

Acute myelogenous leukemia - current recommendations and approaches in molecular-genetic assessment.

Acute myelogenous leukemia is a multi-step hematological malignancy, affecting function, growth, proliferation and cell cycle of myeloid precursors. Overall assessment of patients with the disease requires among everything else, a comprehensive investigation of the genetic basis through various methods such as cytogenetic and molecular-genetic ones. This clarification provides diagnostic refinement and carries prognostic and predictive value in respect of essential therapeutic choices.With this review of the literature, we focus on summarizing the latest recommendations and preferred genetic methods, as well as on emphasizing on their general benefits and limitations. Since none of these methods is actually totipotent, we also aim to shed light over the often-difficult choice of appropriate genetic analyses.

CFTR gene variants as a reason for impaired spermatogenesis: a pilot study and a Meta-analysis of published data.

There is increasing data that IVS8-5T variand and TG repeats could lead to impaired spermatogenesis. To investigate this we performed Sanger sequencing on 50 Bulgarian men with a sperm count below 5 × 106/mL and 20 normal fertile men. Frequencies of the results were compared among the two groups. A meta-analysis was perfomed by using the data for 6,423 patients and 5,834 control subjects, tested for the IVS8-5T polymorphism. One case subject (2.0%) was homozygote for the 5 T/5T variant whereas two (4.0%) were heterozygotes for the 5 T/7T variant. No 5 T alleles were found in the control group. The genotypes of the two groups showed a statistically significant difference (p = 0.04, α < 0.05). Also, the odds ratio was 3.73, but this was unsignificant (p = 0.38). All control subjects had 11 TG repeats and for the test group: 47 (94.0%) men with 11 TG repeats and three (6.00%) with 10 TG repeats. Fisher's test showed no significant difference (p = 0.55). The meta-analysis showed that IVS8-5T variant was a risk factor for impaired spermatogenesis (OR = 2.84, p < 0.05) and this was more prominent for non-European (OR = 4.50, p < 0.05) compared to European (OR = 1.28, p < 0.05) men. The IVS8 - 5 T variant could be associated with disorders of sperm production.

Galectin-3 in patients with atrial fibrillation and restored sinus rhythm.

INTRODUCTION: Cardiac fibrosis is the hallmark of atrial remodeling in atrial fibrillation. Galectin-3 (Gal-3) is a biomarker of fibrosis. It is well studied in heart failure, but the data about its role in atrial fibrillation are sparse. AIM: The aim of the study was to evaluate the levels of Gal-3 in patients with atrial fibrillation after sinus rhythm restoration, to examine the association between this biomarker and other factors for developing atrial fibrillation and to assess its prognostic role. MATERIALS AND METHODS: We included 67 patients (35 male) at the mean age of 67.36±7.25 years, with Gal-3 test after sinus rhythm restoration, a subgroup of participants in placebo-controlled randomized clinical trial of treatment with spironolactone. They were followed up for atrial fibrillation recurrence and hospitalizations. The effect of demographic parameters and other factors on Gal-3 levels were evaluated before and one year after treatment. RESULTS: Mean Gal-3 at baseline was 16.9±6.8 ng/ml. Higher levels of Gal-3 were associated with female gender (р=0.008), increasing age (р=0.005), renal dysfunction (p<0.0001) and gout (р=0.002). Higher thromboembolic risk as assessed by CHA2DS2-VASc score was significantly related to Gal-3. The levels of biomarker did not affect the number of atrial fibrillation recurrences (p=0.9) and hospitalizations. No correlation was found with treatment with spironolactone, antiarrhythmic and antihypertensive drugs. CONCLUSIONS: Higher Gal-3 in atrial fibrillation was associated with female sex, renal dysfunction, and history of gout. The levels of Gal-3 were not related to rhythm control. Treatment with spironolactone did not affect the biomarker of fibrosis Gal-3 in AF patients. Higher Gal-3 was related to high embolic risk.

Association between polymorphic markers human leucocyte antigen-G and tumour necrosis factor alpha and susceptibility to recurrent miscarriages among Bulgarian women.

OBJECTIVE: To analyze the role of 14 base pair (bp) insertion (ins)/deletion (del) and tumour necrosis factor alpha (TNF-α) G/A polymorphisms as risk factors for spontaneous miscarriage in patients with two or more unsuccessful pregnancies and a group of control women with at least two normal live births. MATERIALS AND METHODS: To investigate the role of these mutations, 50 patients with two or more idiopathic recurrent miscarriages and 50 normal fertile women were tested for the presence of human leucocyte antigen-G (HLA-G) 14 bp ins/del and TNF-α -308 G/A variants. The frequencies of the studied polymorphisms were compared between the two groups. RESULTS: Individuals with a history of miscarriages had a significantly higher prevalence of 14 bp insertion alleles compared with control patients (p=0.04). There was also a two times higher relative risk for miscarriage among carriers of this variant. No statistical difference in allele frequencies of the TNF-α -308 G/A polymorphism was established between controls and study patients (p=0.78). CONCLUSION: The 14 bp ins HLA-G variant could be associated with a higher risk for unsuccessful pregnancy according to the results from the study. There is no association between the studied TNF-α -308 GA polymorphism and rate of spontaneous abortions.

Serum expression levels of miR-17, miR-21, and miR-92 as potential biomarkers for recurrence after adjuvant chemotherapy in colon cancer patients.

The present study examined whether miR-17, miR-21, miR-29a, and miR-92 that are dysregulated in colon cancer (CC) can serve as potential predictive markers for relapse of disease after radical surgery and adjuvant chemotherapy. Real-time reverse transcription quantitative polymerase chain reaction was used to measure the expression levels of the miRNAs in serum samples from 37 patients with CC and 7 healthy individuals, tested as a control group. The area under the receiver operating characteristic curve (AUC) was then used to evaluate the predictive performance of the four miRNAs alone or in combination and compare it with carcinoembryonic antigen. The expression of miR-17, miR-21 and miR-92 were significantly higher in serum of patients with disease relapse. The AUCs for miR-17, miR-21, miR-92 for Nx patients were 0.844, 0.948, and 0.935, respectively (p < 0.05). Combining the four miRNAs for stage III patients increased the diagnostic performance, yielding an AUC of 0.881, with a sensitivity of 83.3% and a specificity of 85.7% (p < 0.05). Our study suggests that the expression levels of serum miR-21, miR-17, and miR-92 in patients with CC who underwent radical surgery and adjuvant chemotherapy may have diagnostic value for differentiating between recurred and non-recurred patients.

Expression of Cyclon/CCDC86, a novel nuclear protein, in the hippocampus of adult non-human primates.

Cyclon (cytokine-induced protein with coiled-coil domain), also known as CCDC86 (coiled-coil domain-containing protein 86), is a nuclear protein expressed in lymphocytes in mice where it regulates T-cell responses. Here we show that Cyclon/CCDC86 is expressed in the hippocampus of adult macaque monkeys, including both the hippocampus proper (cornu Ammonis) and the dentate gyrus. Cyclon/CCDC86 was localized to neurons and astrocytes in cornu Ammonis, while in the dentate gyrus the protein was also expressed in immature neurons and a very small fraction of proliferating cells. These results point to a novel expression pattern of Cyclon/CCDC86 beyond its currently known immune cell localization.

Expression and differential response to haloperidol treatment of Cyclon/CCDC86 mRNA in schizophrenia patients.

A gene known as Cyclon (cytokine-induced protein with coiled-coil domain) or CCDC86 (coiled-coil domain-containing protein 86) is known for its expression in leukocytes in mice, where it regulates the immune response. We investigated whether Cyclon/CCDC68 is expressed in leukocytes of schizophrenia patients and whether it might be used as a biological marker for the disease endophenotype segregation. We examined the level of mRNA of Cyclon/CCDC68 in white blood cells obtained from schizophrenia patients in relapse and remission as well as in healthy controls. The mRNA of Cyclon/CCDC68 was expressed by white blood cells of both schizophrenia patients and healthy controls. There was a dichotomous change in the levels of Cyclon/CCDC68 of relapsed patients before and after treatment. High Cyclon/CCDC68 levels were associated with a recent disease and presence of psychotic symptoms, while low levels were associated with a long duration of the disease and an absence of psychotic symptoms. These data indicate that Cyclon/CCDC68 levels correlate with the clinical presentation of relapsed schizophrenia. Cyclon/CCDC68 might be involved in the immune system disturbances observed in schizophrenia.

Correlation between the in vitro antioxidant activity and polyphenol content of aqueous extracts from Bulgarian herbs.

The water phase antioxidant activity of extracts from 23 Bulgarian medicinal plants was studied in relation to their polyphenol content in comparison with mate, black tea, honeybush and rooibos foreign species. Antioxidant activity was measured by the ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) cation radical decolorization assay, and the total polyphenol content was assayed according to the Folin-Ciocalteu method. Five Bulgarian plant extracts exhibited higher antioxidant activity than that of mate, which is 21.7% of all Bulgarian herbs included in this study. These were Alchemilla vulgaris L. (4.79 +/- 0.14 mm), Sambucus ebulus L. (4.03 +/- 0.07 mm), Mentha spicata L. (3.90 +/- 0.03 mm), Fragaria vesca L. (3.74 +/- 0.06 mm), Crataegus monogyna Jacq. (3.63 +/- 0.05 mm). Another eight Bulgarian medicinal plant extracts exhibited an intermediate antioxidant activity - lower than that of mate and higher than that of honeybush, which makes 34.8% of all Bulgarian herbs included in the study. More than half of the herbal extracts included in the present study exhibited antioxidant activity higher than or comparable to the reference foreign plants. A positive correlation (r = 0.92) between antioxidant activity and polyphenol content was found, suggesting that the antioxidant capacity of the aqueous plant extracts is due to a great extent to their polyphenols.