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.

Background: SARS-CoV-2 is a coronavirus that causes a respiratory disease, COVID-19. For COVID-19 testing, real-time PCR is considered gold standard and therefore many commercial SARS-Cov-2 detection kits are available. Objective: The aim of the study is to determine diagnostic values of 10 different commercially available SARS-CoV-2 detection kits, based on their Ct value. Methods: For this study thirty clinical nasopharyngeal samples were collected in ALEA Genetic Center. Twenty four of them were positive, while six were negative and used as a negative control. Positive samples were selected based on the day when first symptoms appeared. RNA was extracted using the same extraction method for all samples. For amplification and comparison of detection kits, the same RT- PCR instrument was used. Results: Accuracy, sensitivity, specificity and Cohen’s kappa coefficient were estimated to evaluate diagnostic values of the tested kits. This study showed that all kits showed 100% specificity. Accuracy, sensitivity and kappa coefficient varied among examined assays. Based on clinical features, LabGunTM COVID-19 Assay by LabGenomics proved to be the most sensitive, the most accurate and most specific. Therefore this assay was used as a reference kit. Conclusion: If things from practice are taken into account, accuracy and reliability of the tested commercial kits can vary compared to those obtained in this study where results were based on ideal functioning of the kits. When choosing the convenient commercial SARS-CoV-2 detection kit using RT-PCR method, many parameters need to be considered.

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.