Rapid screening test of most frequent BRCA1/BRCA2 pathogenic variants in the NGS era.

The average risk of breast cancer in general Slovak population of women is 4-5% and the risk of ovarian cancer is 2%. Probability of breast/ovarian cancer development is higher in individuals carrying a causative germline DNA variant in BRCA1 or BRCA2 gene responsible for hereditary breast/ovarian cancer (HBOC). Although a major proportion of inherited breast/ovarian cancers are due to the mentioned causal mutations, a number of new genes have emerged. Here we describe a rapid, multiplex and comprehensive approach for the detection of pathogenic variants in BRCA1 and BRCA2 genes which most frequently occur in Slovak HBOC population. Analysis comprises the combination of mutation specific methods. Fluorescent PCR amplification followed by fragment analysis for detection of insertions/deletions in exon 11 of BRCA1 gene. Second method is SNaPshot analysis for detection of the most frequent missense and ins/del variants in exons 2, 5, 13, 20 of BRCA1 and exons 11, 23 and 25 of BRCA2 gene. Altogether, we have analyzed 687 samples, 86 (12.5%) in group 1, which fulfilled indication criteria based on the positive family/personal history. Group 2 involved 601 (87.5%) cases, who did not meet the indication criteria and only the screening test was recommended. Using the combined approach, we have identified 47 mutated samples (6.8%), 40 in group 1 (46.5%) and 7 in group 2 (1.1%). However, the presented screening test would not provide complex results of BRCA1/2 gene analysis, it offers testing accessible to a broader spectrum of individuals under the threshold of indication for whole gene analysis. This approach may provide valuable information even in the NGS analysis era.

Comprehensive Genomic Profiling in Predictive Testing of Cancer.

Despite the rapid progress in the field of personalized medicine and the efforts to apply specific treatment strategies to patients based on the presence of pathogenic variants in one, two, or three genes, patient response to the treatment in terms of positive benefit and overall survival remains heterogeneous. However, advances in sequencing and bioinformatics technologies have facilitated the simultaneous examination of somatic variants in tens to thousands of genes in tumor tissue, enabling the determination of personalized management based on the patient's comprehensive genomic profile (CGP). CGP has the potential to enhance clinical decision-making and personalize innovative treatments for individual patients, by providing oncologists with a more comprehensive molecular characterization of tumors. This study aimed to highlight the utility of CGP in routine clinical practice. Here we present three patient cases with various advanced cancer indicated for CGP analysis using a combination of SOPHiA Solid Tumor Solution (STS, 42 genes) for DNA and SOPHiA RNAtarget Oncology Solution (ROS, 45 genes and 17 gene fusions with any random partners) for RNA. We were able to identify actionable genomic alterations in all three cases, thereby presenting valuable information for future management of these patients. This approach has the potential to transform clinical practice and greatly improve patient outcomes in the field of oncology.

Circulating exosomal miRNAs as a promising diagnostic biomarker in cancer.

Cancer belongs to multifactorial diseases characterized by uncontrolled growth and proliferation of abnormal cells. Breast cancer, non-small cell lung cancer, and colorectal cancer are the most frequently diagnosed malignancies with a high mortality rate. These carcinomas typically contain multiple genetically distinct subpopulations of tumor cells leading to tumor heterogeneity, which promotes the aggressiveness of the disease. Early diagnosis is necessary to increase patient progression-free survival. Particularly, miRNAs present in exosomes derived from tumors represent potential biomarkers suitable for early cancer diagnosis. Identification of miRNAs by liquid biopsy enables a personalized approach with the subsequent better clinical management of patients. This review article highlights the potential of circulating exosomal miRNAs in early breast, non-small cell lung, and colorectal cancer diagnosis.

Use of direct sequencing for detection of mutations in the BCR-ABL kinase domain in Slovak patients with chronic myeloid leukemia.

The presence of BCR-ABL oncogene mutations in patients with chronic myeloid leukemia (CML) may be responsible for the failure of tyrosine kinase inhibitor (TKI) treatment. The aim of the study was to evaluate the frequency of BCR-ABL gene mutations in patients with CML treated with tyrosine kinase inhibitors. Our lab received 64 samples (34 women, 30 men) from patients with CML who failed or had suboptimal response to TKI treatment. The mutation analysis was performed in 61 patients with CML, 3 patients could not be tested because of inadequate RNA quality. An 866 base pair fragment containing the ABL kinase domain was amplified in a seminested RT (reverse transcriptase)-PCR and then sequenced using Applied Biosystems BigDye Terminator chemistry with two pairs of primers. We analyzed 61 patients with CML, 11 mutations were detected in 13 (21%) patients and SNP (single nucleotide polymorphism) in 6 patients (10%). In addition to 9 point mutations (G250E / F317L, F359V, L387M, Y253H, M388L, M244V, T315I, D276G), 35 bp insertion between exons 8 and 9 and deletion exon 7 were detected. Our results demonstrate that direct sequencing is suitable for routine clinical monitoring patients with CML and may be useful for optimizing therapy.

DNA analysis of Y chromosomal AZF region in Slovak population with fertility disorders.

BACKGROUND: The Y chromosome is characterized by a low number of functional genes, relatively high number of repetitive sequences and the ability of recombination purely by short arms of telomeres PAR1 and PAR2. The long arm contains an AZF region with genes participating in spermatogenesis. Microdeletions of three subregions, namely AZFa,b,c and their mutual combinations are responsible for male infertility and the resulting azoospermia and oligospermia. OBJECTIVES: The aim of this study based on evaluating 822 patients during a period of ten years was to analyse types of microdeletions in men with fertility disorders in Slovakia. METHODS: For detecting the microdeletions in Y-chromosomal AZF region and for identifying the Y-specific sequences we used PCR while using three different sets of sY sequences. REPORTS: We reported 38 cases of deletions in AZF region, namely 18 cases when using the first set of sequences, 12 cases when using the second set, and finally 8 cases when using the third set. When using the last set of sequences according to the European Academy of Andrology and European Molecular Genetics Quality Network, we detected deletions only in patients with azoospermia. In addition to deletions in each of AZF a,b,c subregions we recorded also a complete deletion of the whole AZF region. In the AZFa subregion, we recorded a deletion of sequence sY86. CONCLUSION: The study has confirmed that the detection of microdeletions of AZF region is significant from the diagnostic and prognostic views (Tab. 5, Ref. 21). Full Text in free PDF www.bmj.sk.

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients.

Colorectal carcinoma (CRC) represents a serious problem worldwide: in the Slovak republic are diagnosed about 2600 new CRC cases annually and its incidence is increasing. Colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of CRC or changing the existing therapeutic procedures. Recent progresses have been made in understanding of EGFR pathway involved in CRC carcinogenesis, especially the role of Ras protein. Mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (EGFR) inhibitor therapies, such as Panitumumab and Cetuximab. Consequently, screening for KRAS mutations status may be used as a prognostic marker, because the CRC patients with KRAS positive tumors have a worse prognosis. The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFPE) tissues DNA. We applied Real Time PCR analysis (TheraScreen KRAS Mutation Test Kit) and sequencing analysis (optimised for the analysis of FFPE tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of DNA isolated from 25 colorectal FFPE tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). Our results demonstrate that the Real Time PCR analysis can be used for detection of somatic KRAS mutations in FFPE clinical samples. However, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for mutations status screening, but with care of method sensitivity.

[Electrophysiologic and electrocardiographic aspects of the transplanted heart]. / Specifické elektrofyziologické a elektrokardiografické aspekty transplantovaného srdca.

Specific electrophysiological properties of transplanted heart are caused mainly by complex surgery, suppression of autonomic innervation, and allograft rejection. Interpretation of ECG may be difficult because of two P waves. The other most prevalent ECG abnormalities are: incomplete or complete right bundle branch block, shift of QRS axis to the left, shorter QT interval, decreased precordial voltage. The influence of denervation is apparent in: higher basic heart rate, chronotropic dysfunction, different responsiveness to various cardiac drugs, susceptibility to fatal ventricular arrhythmias. The prevalence of bradyarrhythmias and tachyarrhythmias is higher than in other population. Many of them correlate with acute or chronic cardiac rejection. It is desirable to utilize specific electrophysiological properties for noninvasive detection of allograft rejection.

[Diagnostic and therapeutic potential of transesophageal cardiac pacing in the management of patients with arrhythmias]. / Diagnostický a terapeutický potenciál transezofágovej kardiostimulácie pre management pacientov s arytmiami.

BACKGROUND: Transoesophageal cardiostimulation is a semiinvasive method of stimulation of atrii enabling the performance of the programmed atrial stimulation without the inevitability of an invasive vascular approach. This method was used in 124 patients with the following indication spectrum. Diagnostic indications: total 82%, paroxysmal supraventricular tachycardia (SVT), and WPW sy-22%, tachycardia with wide QRS-complex-8%. SSS syndrome and bradycardia-20%, sycopes and collapses with unclear etiology-13%, palpitations-11%, control of antiarrhythmic therapy-4%, and other states-6%. Therapeutic indications: total-18%, versions of paroxysmal SVT and flutter of atrii. RESULTS: The patients with SVT were assumed to develop the arrhythmogenic mechanism--AV nodal re-entry tachycardia in 80%, orthodrome AV-re-entry tachycardia in 30%, and flutter of atrii in 20%. All patients with WPW-syndrome were stratified by the use of this method. The origin of this state from ventricular arrhythmia was verified in 40% of patients with tachycardia with a wide QRS complex. In coincidence with other indications, the diagnostic benefit of transoesophageal cardiostimulation was evaluated as follows: syncopes-68%, palpitations-64%, syndrome SSS and bradycardia-48%. The therapeutic indication of SVT version and flutter of atrii, was totally successful in 40%, partly successful in 45% and unsuccessful in 15% of patients. CONCLUSION: Transoesophageal cardiostimulation has contributed to the assessment of the diagnosis in 69% of patients and has acutely managed arrhythmia in 85% of cases. According to our experience, this method is effective in the initial management of patients with arrhythmia. Its low technical and economic demands make its wider utilisation appropriate in clinical practice of internal medicine. (Tab. 4, Fig. 9, Ref. 22).

[Arrhythmogenic right ventricular dysplasia]. / Arytmogénna dysplázia pravej komory.

Arrhythmogenic dysplasia of the right ventricle as a nosological entity was described relatively recently. However, at present, it is gradually being diagnosed more frequently. The authors describe the typical clinical picture of this disease in one of several patients with this disease, who were hospitalised at SUSCH. The authors describe the diagnostical value of individual findings. They indicate the importance of knowledge of individual diagnostical criteria in order to be able to recognize this disease and distinguish it from idiopathic ventricular tachycardia, the prognosis of which is generally better and also the therapeutical approach less aggressive. (Fig. 6, Ref. 29).

[Detection and diagnosis of hereditary monogenic neurologic diseases in Slovakia]. / Záchytnost a diagnostika dedicných monogénne podmienených neurologických ochorení na Slovensku.

Authors present a clinical symptoms recapitulation of the most important monogenic hereditary neuromuscular diseases, their molecular-genetic causes and the possibilities of diagnostic on the level of DNA analysis. Low detectability of these pathologic states in Slovak republic is stressed and possible causes of this state are analyzed. (Ref. 10.)

[Indirect diagnosis of type I neurofibromatosis using RFLP in Slovak families]. / Nepriama diagnostika neurofibromatózy typ 1 pomocou RFLP v slovenských rodinách.

Neurofibromatosis type I clinical diagnosis confirmation as well as antenatal diagnostics of the disease are recently provided by molecular genetics. The authors analyze 17 Slovak families with multiple NFI incidence, in whom the detection of mutated gene transfer was performed using indirect diagnostics-bound with of restrictive fragments length polymorphism RFLP. With the help of PCR 7 polymorphic sequencies were amplified and subsequently broken with restrictive endonucleases localized close to the neurofibrin gene. The system informative capacity was comparable with the results of other Caucasian population studies. Although direct detection of mutation is the perspective of the diagnostics, binding analysis in informative families with multiple incidence of the disease provides reliable and cheaper possibility of NFI diagnostic on the level of DNA analysis. (Tab. 1, Fig. 3, Ref. 26.)

[Diagnosis of Duchenne and Becker muscular dystrophy in Slovak patients using multiplex polymerase chain reaction]. / Diagnostika Duchenneovej a Beckerovej muskulárnej dystrofie pomocou mnohonásobnej polymerázovej retazovej reakcie u slovenských pacientov.

The authors of the paper describe the diagnostic method of deletion in the dystrophin gene by means of an improved variant of the polymerase chain reaction--so called multiplex PCR. The authors analyzed a group of 66 patients with developed clinical symptoms of the disease. The deletion screening included 22 exones of the dystrophine gene and it was performed in 5 multiplex PCR reactions. 20 patients yielded a verified deletion which was pre-assessed by Southern's hybridization. The relative simplicity of multiplex PCR which does not require the use of radioisotopes, its low time and financial needs, make this method to represents an appropriate alternative of Southern's hybridization in the assessment of deletion of the dystrophine gene. (Fig. 1, Ref. 19.)

Reduction of symptoms from LVOT tachycardia following inadvertent fast pathway ablation.

VT was mapped to above the aortic valve in a young patient with troublesome palpitations. A single 15-second RF application was inadvertently delivered to a reference His catheter producing permanent first-degree heart block. The patient has been completely asymptomatic since.

Radiofrequency ablation of haemodynamically unstable ventricular tachycardia after myocardial infarction.

OBJECTIVE: To determine whether radiofrequency (RF) ablation might have a role in haemodynamically unstable ventricular tachycardia. METHODS: 10 patients with a history of ventricular tachycardia producing haemodynamic collapse in whom drug treatment had failed and device therapy was rejected underwent RF ablation of ventricular tachycardia in sinus rhythm. The arrhythmogenic zone was defined on the basis of abnormal systolic movement, the presence of fragmentation (low amplitude, prolonged multiphasic electrograms), and pace mapping. RF lesions were delivered in power mode in linear fashion within the defined arrhythmogenic zone. RESULTS: Success (no ventricular tachycardia inducible postablation or at retest) was achieved in six patients, possible success (a different ventricular tachycardia inducible at more aggressive stimulation) in three. In one patient, the procedure was abandoned because of poor catheter stability. There were no clinical events during a mean (SD) follow up period of 23 (10) months in any of the nine patients defined as definite or possible successes. CONCLUSIONS: RF ablation for addressing haemodynamically unstable ventricular tachycardia opens the door for the wider use of catheter ablation for treating this arrhythmia.

PCR based diagnosis of 21-hydroxylase gene defects in Slovak patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyse 21-hydroxylase gene for 8 most common mutations in patients with salt-wasting type of congenital adrenal hyperplasia. METHODS: Allele specific PCR performed on 8 salt-wasting CAH patients and their 23 healthy relatives. RESULTS: Two patients were homozygous for 8 bp deletion in exon 3, while 6 patients were homozygous for intron 2 splice mutation. Mutant allele for splice mutation was found also in both parents of patients with this type of mutation. CONCLUSIONS: These preliminary results show that only two mutations, 8 bp deletion in exon 3 and splice mutation in intron 2, were present in this group of Slovak patients with salt-wasting type of congenital adrenal hyperplasia.