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.

Molecular screening for fragile X syndrome in children with unexplained intellectual disability and/or autistic behaviour.

INTRODUCTION: Fragile X syndrome (FXS, OMIM #300624) is the most common inherited form of intellectual disability and the leading monogenic cause of autism.

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.

16p11.2 Duplication Syndrome - a Case Report.

16p11.2 duplication syndrome is a rare disorder, often associated with intellectual disability, attention deficit, hyperactivity disorder, and a predisposition to epilepsy and schizophrenia. There are no specific dysmorphic features for this genetic condition, but micro-cephaly, micrognathia and hypertelorism could be present. We report a case of 16p11.2 duplication syndrome which has the typical clinical presentation - slight facial dysmorphism, impaired intellectual development, and autistic behavior. Whole-exome sequencing was performed, but no pathogenic or likely pathogenic mutations were identified. Array comparative genomic hybridization analysis established the diagnosis of 16p11.2 duplication syndrome, which illustrates the importance of this method when diagnosing children with unexplained intellectual disability.

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.

Comparison between thrombophilic gene polymorphisms among high risk patients.

INTRODUCTION: The purpose of this study was to compare the role of the thrombophilic variants among two groups of high risk patients with vascular disorders and recurrent pregnancy loss. METHODS: 200 patients, including 76 with thrombotic accidents and 124 with two or more idiopathic recurrent miscarriage during the first trimester, were tested for the presence of Factor V (F V) Leiden G1691A, Factor II (F II) G20210A, plasminogen activator inhibitor (PAI) 4G/5G, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms using Real time polymerase chain reaction (RT - PCR) in the Laboratory of Medical Genetics, Varna, Bulgaria between June 2016 and May 2019. Frequencies of thrombophilic gene polymorphisms were compared among the two populations and to the expected genotype frequencies. RESULTS: Individuals with a history of vascular disorders had a significantly higher frequency of F V Leiden variant compared to women with recurrent miscariage. There was no statistical difference between the analyzed patients for the other three thrombophilic polymorphisms. The allelic frequencies and the expected genotype frequencies of the F V, F II and MTHFR polymorphisms were calculated according to Hardy-Weinberg equilibrium. The percentages of the homozygotes for F V and F II were higher than expected in the two groups of patients. For the MTHFR there was no difference. CONCLUSION: F V Leiden remains the strongest risk factor for vascular disorders and recurrent pregnancy loss. Screening for this variant should be recommended to patients with thrombotic accidents and women with repeated miscarriage. The role of F II, PAI and MTHFR remains controversial.