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D. Pećar

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Human mitochondrial genes MT-ATP6 and MT-ATP8 encode the subunits 6 and 8, respectively, of ATP synthase, a vital protein Complex V intricately involved in oxidative phosphorylation and ATP metabolism. This enzyme produces ATP from ADP in the mitochondrial matrix utilizing energy provided by the proton electrochemical gradient. Pathogenic mutations within these genes have been linked to various syndromes such as NARP syndrome, Leigh syndrome, mitochondrial myopathy with reversible cytochrome C oxidase deficiency, and progressive spastic paraparesis, among others. In our investigation, we sequenced 24 complete human mitochondrial genomes of healthy adult individuals from Bosnia and Herzegovina, each representing unique maternal lineage. Employing the Illumina MiSeq NGS platform and the Nextera XT DNA library preparation protocol, we obtained raw NGS reads. Subsequent analysis utilizing SAMtools enabled the identification of genetic variants within the MT-ATP6 and MT-ATP8 genes. We identified a total of 11 SNPs, including three in MT-ATP8 and eight in MT-ATP6, with none of them being associated with any mitochondrial diseases or conditions. Our results align well with previously reported genome variation data for European populations and set the groundwork for future mtDNA analysis for clinical purposes in Bosnia and Herzegovina.

Abstract Background Almost 50% of NSCLC patients who initially show a successful response to tyrosine kinase inhibitors targeted therapy (TKI therapy) eventually develop acquired EGFR T790M mutation. The T790M secondary mutation can cause resistance to the targeted therapy and disease relapse. Since this mutation can be present at very low frequencies in liquid biopsy samples, droplet digital PCR (ddPCR), due to its high sensitivity, has opened the possibility for minimally invasive monitoring of the disease during TKI targeted therapy. Materials and methods For this study, a total of 45 plasma samples from NSCLC patients with previously detected EGFR-activating mutations were analyzed. Extracted circulating free DNA was amplified and examined for the presence of T790M mutation using ddPCR technology. For the data analysis, QuantaSoft Software was used. Results Of 45 tested plasma samples, a total of 14 samples were identified as positive for the T790M mutation. The same samples eventually showed the presence of T790M mutation in FFPE. Droplet digital PCR showed its great advantage in high sensitivity detection of rare allele variants. Our ddPCR assay detected T790M mutant allele in frequencies from 0.1%. The average number of droplets generated by ddPCR was 9571. Conclusion Monitoring of the T790M mutation has an important role in the examination of the effects of the prescribed TKI therapy. Since monitoring of potential changes during TKI therapy requires repeated sampling, our results showed that ddPCR technology has made it possible to use liquid biopsy as an adequate minimally invasive alternative for single nucleotide polymorphisms (SNP) detection.

Introduction: COVID-19 has been a major focus of scientific research since early 2020. Due to its societal, economic, and clinical impact worldwide, research efforts aimed, among other questions, to address the effect of host genetics in susceptibility and severity of COVID-19. Methods: We, therefore, performed next-generation sequencing of coding and regulatory regions of 16 human genes, involved in maintenance of the immune system or encoding receptors for viral entry into the host cells, in a subset of 60 COVID-19 patients from the General Hospital Tešanj, Bosnia and Herzegovina, classified into three groups of clinical conditions of different severity (“mild,” “moderate,” and “severe”). Results: We confirmed that the male sex and older age are risk factors for severe clinical picture and identified 13 variants on seven genes (CD55, IL1B, IL4, IRF7, DDX58, TMPRSS2, and ACE2) with potential functional significance, either as genetic markers of modulated susceptibility to SARS-CoV-2 infection or modifiers of the infection severity. Our results include variants reported for the first time as potentially associated with COVID-19, but further research and larger patient cohorts are required to confirm their effect. Discussion: Such studies, focused on candidate genes and/or variants, have a potential to answer the questions regarding the effect of human genetic makeup on the expected infection outcome. In addition, loci we identified here were previously reported to have clinical significance in other diseases and viral infections, thus confirming a general, broader significance of COVID-19-related research results following the end of the pandemic period.

Background: All viral genomes, including the SARS-CoV-2 virus, mutate over time, and some of these mutations can affect the characteristics of the virus, such as the ease of spread, the severity of the patient’s clinical picture, or the effect of vaccines, therapeutic drugs, diagnostic tools or other measures of public health and social protection. Because of all the above, it is imperative to carry out continuous sequencing of this pathogen. Objective: The main goal of this research was to obtain the highest quality genomic sequences of the SARS-CoV-2 virus, to compare the obtained sequences with the reference Wuhan-Hu-1 sequence and to obtain a high-quality genomic alignment in order to reconstruct the appropriate phylogenetic tree. Methods: For the purposes of this research, a next-generation semiconductor sequencing method was chosen. In this research, a total of 47 samples of nasopharyngeal and oropharyngeal swabs from patients from the human population of Bosnia and Herzegovina with a clinical diagnosis of COVID-19 were collected. Results: In the processed 47 samples, there are several monophyletic groups on the constructed phylogenetic tree, of which one sample belongs to the same monophyletic group as the Wuhan-Hu-1 reference sequence. Conclusion: The greater number of samples is needed for a more comprehensive approach. Therefore, the results of this research can act as a guideline for the design of effective measures and strategies in order to solve problems regarding future pandemics as efficiently as possible.

Nejla Muhovic - Pasic, Mirela Kahvic, Selma Karup, D. Pećar, E. Kandić, L. Salihefendić, Rijad Konjhodžić

Background: Human papillomavirus is a sexually transmitted infection and it is estimated that 75% of all women have been exposed to HPV infection in a certain period of life. High-risk types of HPV are considered to be one of the major causes of cervical cancer and its precursor intraepithelial neoplasia. Objective: The aim of this study was to investigate the degree of HPV infections and to provide more data on HPV genotype distribution among women in Bosnia and Herzegovina (B&H). Methods: Number of 375 samples were collected from different polyclinics in Sarajevo and were analyzed by Alea Genetic Center using Genomed f-HPV typing™ multiplex Fluorescent PCR kit for human papillomavirus genotyping. DNA required for this method is extracted from cervical swabs and amplified using a multiplex PCR reaction containing a set of 16 fluorescently labeled primers that recognize 16 HPV types. 14 HPV types are classified as high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and two are low-risk (6 and 11) HPV types. Results: Results showed that in the years 2018, 2019, and 2021, HPV type 16 is predominant causing the high-risk factor for CIN1, CIN2, CIN3, and cervical cancer development. HPV 18 infection rates decreased during the last four years of study. HPV 6 infection rates increased during that period of time. Conclusion: HPV 16 and HPV 18 are almost completely preventable by vaccination implying that the number of diagnosed cervical cancers in B&H could be much lower in the next decades if the HPV vaccination routine immunization program starts soon.

The goal of this part of the study was to optimize the sequencing procedure for 16 human genes and their regulatory regions that might be associated with differential immunological response to COVID-19. The study was performed on 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina, categorized into three groups of mild, moderate, and severe clinical manifestation, based on the diagnosis by the residential physician. Target coding sequences and their regulatory regions were amplified for the following genes: HLA-A, HLA-B, HLA-C, ACE2, IL-6, IL-4, TMPRSS2, IFITM3, IL-12, RIG-I/DDX58, IRF-7, IRF-9, IL-1B, IL-1A, CD55, and TNF-α. DNA was isolated from the whole blood samples stored at -20°C for six months using QIAamp® DNA Mini Kit according to manufacturer’s instructions. Since NGS analysis of target genomic regions was performed on the Ion Torrent GeneStudio™ S5 platforms, libraries were prepared using Ion AmpliSeq™ Library Kit Plus according to manufacturer’s instructions in a protocol optimized for low-quality DNA. Due to dissatisfactory sequencing results, further protocol optimization steps were employed through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool which showed poorer amplification results. In the end, 36 samples produced optimal results, while the remaining 24 samples will be re-sequenced following repeated sample collection and DNA isolation, accompanied by additional protocol modifications.

Azra Hajdarević, L. Salihefendić, Rijad Konjhodžić, Adis Kandic, I. Čeko, D. Pećar, Muhamed Curic, Amina Kurtovic - Kozaric

Cancer is an abnormal proliferation of cells that is characterized by the presence of genomic alterations including DNA mutations, deletions, insertions, translocations, inversions, and others. KRAS gene is one of the most mutated genes across different cancer types. The most common mutations in KRAS are found in codons 12 and 13 of KRAS protein, which are associated with a lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapy. This study assessed and compared the performance between two diagnostic methods: droplet digital PCR (ddPCR) and next generation sequencing (NGS). The main goal was to determine KRAS G12 / G13 mutant allele fraction using NGS and to compare the accuracy toddPCR. A total of 28 samples of non – small cell lung cancer (NSCLC) and colorectal cancer (CRC) were analyzed using ddPCR and NGS methods. Our results show that even though both methods exhibited high rate of concordance and correlation, the study proved that ddPCR is more superior when it comes to detecting low frequency mutations. Even though strong correlation was observed, based on the values obtained, we concluded that ddPCR is more accurate, reliable, and sensitive in comparison with NGS.

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

Next Generation Sequencing (NGS) has become powerful tool in molecular oncology. It allows multiparallel targeted sequencing that enables comprehensive assessment of tumor heterogeneity. Detection of mutations in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) defines patients diagnosis, therapy and prognosis. Multiple genes, their somatic mutations to be precise, carry different degrees of importance for any of these stages. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which was used in this study, allows detection of hotspot mutations on 22 genes in a single reaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequency mutation detection and it has been used as a validation method for mutations that were detected with NGS. It has high sensitivity and enables accurate detection of mutant allele in a background of abundant wild type alleles. For this study 35 samples of CRC and NSCLC were sequenced and same samples were analysed on ddPCR for KRAS, NRAS, EGFR and BRAF genes. All processed samples were successfully sequenced and had average base coverage >500X. NGS sequencing proved itself to be cost effective, has shorter turnaround time and is highly sensitive. Out of 35 samples, 25 had genetic alterations, while 10 samples are reported as wild type but were still tested on ddPCR as controls. In three samples low frequency somatic mutations were detected by NGS and verified using ddPCR, which leads us to conclusion that ddPCR is a good tool for verification of somatic mutations in CRC and NSCLC.

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