Identification of human genetic variants modulating the course of COVID-19 infection with importance in other viral infections

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

DNA polymorphisms detected in MT-ATP6 and MT-ATP8 genes in the residents of Sarajevo Canton

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

Haplogroup Prediction Using Y-Chromosomal Short Tandem Repeats in the General Population of Bosnia and Herzegovina

Human Y-chromosomal haplogroups are an important tool used in population genetics and forensic genetics. A conventional method used for Y haplogroup assignment is based on a set of Y-single nucleotide polymorphism (SNP) markers deployed, which exploits the low mutation rate nature of these markers. Y chromosome haplogroups can be successfully predicted from Y-short tandem repeat (STR) markers using different software packages, and this method gained much attention recently due to its labor-, time-, and cost-effectiveness. The present study was based on the analysis of a total of 480 adult male buccal swab samples collected from different regions of Bosnia and Herzegovina. Y haplogroup prediction was performed using Whit Athey’s Haplogroup Predictor, based on haplotype data on 23 Y-STR markers contained within the PowerPlex® Y23 kit. The results revealed the existence of 14 different haplogroups, with I2a, R1a, and E1b1b being the most prevalent with frequencies of 43.13, 14.79, and 14.58%, respectively. Compared to the previously published studies on Bosnian-Herzegovinian population based on Y-SNP and Y-STR data, this study represents an upgrade of molecular genetic data with a significantly larger number of samples, thus offering more accurate results and higher probability of detecting rare haplogroups

Forensic DNA databasesin Western Balkan region:retrospectives, perspectives, and initiatives

The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a ‘regional supplement’ to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations

Identification of the mitochondrial DNA control region polymorphisms in population of Bosnia and Hercegovina and the development of the protocol for their forensic application

Cilj rada: utvrđivanje žarišta SNP-ova na HV-1 regiji u populaciji Bosne i Hercegovine, utvrđivanje zastupljenosti alternativnih inačica SNP-ova na utvrđenim žarištima, utvrđivanje zastupljenosti tipova mtDNA unutar procesiranih uzoraka, i kreiranje protokola za detekciju istih putem metode jednobazne ekstenzije. ----- Materijali i metode: iz uzorka od 300 bukalnih sluznica uzetih od nesrodnih pojedinaca izolirana je DNA, na kojoj je amplificirana HV-1 regija korištenjem univerzalnih početnica. Amplificirana HV-1 regija je prvo kvantificirana, a potom i sekvencirana Sangerovom metodom na automatiziranom DNA sekvenceru. Oslanjajući se na tako prikupljene podatke, kreirane su početnice specifične za odabrana žarišta, i provedena je njihova pojedinačna detekcija metodom jednobazne ekstenzije. Nakon toga, kreirane početnice su pomiješane u multipleks reakcije, koje su provedene prvo na individualnim uzorcima, te potom na miješanom uzorcima. U istraživanju provedenom u okviru ove disertacije korišteni su ABI BigDye v1.1, ABI SNAPShot i ABI Quantifiler kitovi, a strojna analiza je napravljena koristeći ABI 3130 Genetic Analyzer i ABI 7700 Sequence Detection System instrumente. ----- Rezultati: analizom elektroferograma sekvenciranja utvrđena su žarišta pojavljivanja SNP-ova, i izračunate zastupljenosti alternativnih inačica SNP-ova na njima. Dobivene sekvence HV-1 regije su podijeljene u 33 tipa mtDNA. Za sve tipove izračunate su vrijednosti GGIP (Gornje Granice Intervala Pouzdanosti), i LR (Likelihood Ratio). Rezultati analize jednobaznom ekstenzijom pokazali su se u potpunosti podudarni s rezultatima sekvenciranja po Sangeru, kako u reakcijama s pojedinačnim početnicama, tako i u multipleks reakcijama. Analize miješanih uzoraka DNA jasno su tipizirale oba tipa mtDNA prisutnih u mješavini. ----- Zaključci: na osnovu dobivenih rezultata, a u skladu s postavljenim ciljevima ovog istraživanja i njihove usporedbe s dosadašnjim istraživanjima, dolazi se do sljedećih zaključaka: izdvojeno je petnaest SNP žarišta čiji lokusi i zastupljenosti alternativne inačice SNP-a tvore jedinstvenu populacijsku studiju, što je izraženo različitim izračunatim vrijednostima statističke signifikantnosti podudaranja unutar kreirane studije naspram sličnih studija iz literature. Kreirana metoda detekcije profiliranja SNP-ova na utvrđenim žarištima jednobaznom ekstenzijom pokazala se funkcionalnom, a njeni rezultati u potpunosti podudarni s rezultatima sekvenciranja. Također, kreirana metoda detekcije jednobaznom ekstenzijom pokazala je visoku osjetljivost u tipiziranju miješanih uzoraka, području nepokrivenom metodom sekvenciranja po Sangeru.Aim of this study was to establish locations of the SNP hotspots in the HV 1 region, their alternative allele frequencies, as well as creation of a protocol for their typing based on the single base extension (SBE). ----- Materials and Methods: DNA was extracted from buccal swabs taken from 300 unrelated individuals. Extracted DNA was first quantified using RT-PCR, and then amplified at the HV-1 region of the mtDNA with adequate primers. Resulting amplicons were sequenced by Sanger sequencing using an automated DNA sequencer. Relying on the data thus acquired, primers specific for the chosen hotspots were designed, and samples were typed using SBE method for each SNP in single reactions. After that, designed primers were mixed in multiplex reaction mixes, and samples were typed with those. Created mixed samples from two different DNAs were tested with both singleplex and multiplex reactions. ----- Results: SNP hotspots and their alternative allele frequencies were determined. Taking these in consideration 33 mtDNA types were observed, with Upper Confidence Interval values and Likelihood Ratios calculated for each type. Results obtained from SBE typing protocol of the samples were identical to Sanger sequencing, both in singleplex and multiplex reactions. SBE protocol successfully typed mixed DNA samples. ----- Conclusion: Taking into consideration both the loci and the alternative allele frequencies of selected 15 SNPs, it can be concluded that they make a unique population study, which is shown with comparing calculations of the given mtDNA profile uniqueness using this study data and the data from studies found in the literature. Chosen SNP typing by the SBE protocol has shown itself functional, with results absolutely matching those acquired with Sanger sequencing. Furthermore, SBE protocol has shown high sensitivity in typing mixed DNA samples

Identification of the mitochondrial DNA control region polymorphisms in population of Bosnia and Hercegovina and the development of the protocol for their forensic application

Cilj rada: utvrđivanje žarišta SNP-ova na HV-1 regiji u populaciji Bosne i Hercegovine, utvrđivanje zastupljenosti alternativnih inačica SNP-ova na utvrđenim žarištima, utvrđivanje zastupljenosti tipova mtDNA unutar procesiranih uzoraka, i kreiranje protokola za detekciju istih putem metode jednobazne ekstenzije. ----- Materijali i metode: iz uzorka od 300 bukalnih sluznica uzetih od nesrodnih pojedinaca izolirana je DNA, na kojoj je amplificirana HV-1 regija korištenjem univerzalnih početnica. Amplificirana HV-1 regija je prvo kvantificirana, a potom i sekvencirana Sangerovom metodom na automatiziranom DNA sekvenceru. Oslanjajući se na tako prikupljene podatke, kreirane su početnice specifične za odabrana žarišta, i provedena je njihova pojedinačna detekcija metodom jednobazne ekstenzije. Nakon toga, kreirane početnice su pomiješane u multipleks reakcije, koje su provedene prvo na individualnim uzorcima, te potom na miješanom uzorcima. U istraživanju provedenom u okviru ove disertacije korišteni su ABI BigDye v1.1, ABI SNAPShot i ABI Quantifiler kitovi, a strojna analiza je napravljena koristeći ABI 3130 Genetic Analyzer i ABI 7700 Sequence Detection System instrumente. ----- Rezultati: analizom elektroferograma sekvenciranja utvrđena su žarišta pojavljivanja SNP-ova, i izračunate zastupljenosti alternativnih inačica SNP-ova na njima. Dobivene sekvence HV-1 regije su podijeljene u 33 tipa mtDNA. Za sve tipove izračunate su vrijednosti GGIP (Gornje Granice Intervala Pouzdanosti), i LR (Likelihood Ratio). Rezultati analize jednobaznom ekstenzijom pokazali su se u potpunosti podudarni s rezultatima sekvenciranja po Sangeru, kako u reakcijama s pojedinačnim početnicama, tako i u multipleks reakcijama. Analize miješanih uzoraka DNA jasno su tipizirale oba tipa mtDNA prisutnih u mješavini. ----- Zaključci: na osnovu dobivenih rezultata, a u skladu s postavljenim ciljevima ovog istraživanja i njihove usporedbe s dosadašnjim istraživanjima, dolazi se do sljedećih zaključaka: izdvojeno je petnaest SNP žarišta čiji lokusi i zastupljenosti alternativne inačice SNP-a tvore jedinstvenu populacijsku studiju, što je izraženo različitim izračunatim vrijednostima statističke signifikantnosti podudaranja unutar kreirane studije naspram sličnih studija iz literature. Kreirana metoda detekcije profiliranja SNP-ova na utvrđenim žarištima jednobaznom ekstenzijom pokazala se funkcionalnom, a njeni rezultati u potpunosti podudarni s rezultatima sekvenciranja. Također, kreirana metoda detekcije jednobaznom ekstenzijom pokazala je visoku osjetljivost u tipiziranju miješanih uzoraka, području nepokrivenom metodom sekvenciranja po Sangeru.Aim of this study was to establish locations of the SNP hotspots in the HV 1 region, their alternative allele frequencies, as well as creation of a protocol for their typing based on the single base extension (SBE). ----- Materials and Methods: DNA was extracted from buccal swabs taken from 300 unrelated individuals. Extracted DNA was first quantified using RT-PCR, and then amplified at the HV-1 region of the mtDNA with adequate primers. Resulting amplicons were sequenced by Sanger sequencing using an automated DNA sequencer. Relying on the data thus acquired, primers specific for the chosen hotspots were designed, and samples were typed using SBE method for each SNP in single reactions. After that, designed primers were mixed in multiplex reaction mixes, and samples were typed with those. Created mixed samples from two different DNAs were tested with both singleplex and multiplex reactions. ----- Results: SNP hotspots and their alternative allele frequencies were determined. Taking these in consideration 33 mtDNA types were observed, with Upper Confidence Interval values and Likelihood Ratios calculated for each type. Results obtained from SBE typing protocol of the samples were identical to Sanger sequencing, both in singleplex and multiplex reactions. SBE protocol successfully typed mixed DNA samples. ----- Conclusion: Taking into consideration both the loci and the alternative allele frequencies of selected 15 SNPs, it can be concluded that they make a unique population study, which is shown with comparing calculations of the given mtDNA profile uniqueness using this study data and the data from studies found in the literature. Chosen SNP typing by the SBE protocol has shown itself functional, with results absolutely matching those acquired with Sanger sequencing. Furthermore, SBE protocol has shown high sensitivity in typing mixed DNA samples

Identifikacija polimorfizama kontrolne regije mitohondrijske DNA u populaciji Bosne i Hercegovine i razvoj protokola za njihovu forenzičku primjenu [Identification of the mitochondrial DNA control region polymorphisms in population of Bosnia and Hercegovina and the development of the protocol for their forensic application]

Aim of this study was to establish locations of the SNP hotspots in the HV 1 region, their alternative allele frequencies, as well as creation of a protocol for their typing based on the single base extension (SBE). ----- Materials and Methods: DNA was extracted from buccal swabs taken from 300 unrelated individuals. Extracted DNA was first quantified using RT-PCR, and then amplified at the HV-1 region of the mtDNA with adequate primers. Resulting amplicons were sequenced by Sanger sequencing using an automated DNA sequencer. Relying on the data thus acquired, primers specific for the chosen hotspots were designed, and samples were typed using SBE method for each SNP in single reactions. After that, designed primers were mixed in multiplex reaction mixes, and samples were typed with those. Created mixed samples from two different DNAs were tested with both singleplex and multiplex reactions. ----- Results: SNP hotspots and their alternative allele frequencies were determined. Taking these in consideration 33 mtDNA types were observed, with Upper Confidence Interval values and Likelihood Ratios calculated for each type. Results obtained from SBE typing protocol of the samples were identical to Sanger sequencing, both in singleplex and multiplex reactions. SBE protocol successfully typed mixed DNA samples. ----- Conclusion: Taking into consideration both the loci and the alternative allele frequencies of selected 15 SNPs, it can be concluded that they make a unique population study, which is shown with comparing calculations of the given mtDNA profile uniqueness using this study data and the data from studies found in the literature. Chosen SNP typing by the SBE protocol has shown itself functional, with results absolutely matching those acquired with Sanger sequencing. Furthermore, SBE protocol has shown high sensitivity in typing mixed DNA samples

Identification of the mitochondrial DNA control region polymorphisms in population of Bosnia and Hercegovina and the development of the protocol for their forensic application

Cilj rada: utvrđivanje žarišta SNP-ova na HV-1 regiji u populaciji Bosne i Hercegovine, utvrđivanje zastupljenosti alternativnih inačica SNP-ova na utvrđenim žarištima, utvrđivanje zastupljenosti tipova mtDNA unutar procesiranih uzoraka, i kreiranje protokola za detekciju istih putem metode jednobazne ekstenzije. ----- Materijali i metode: iz uzorka od 300 bukalnih sluznica uzetih od nesrodnih pojedinaca izolirana je DNA, na kojoj je amplificirana HV-1 regija korištenjem univerzalnih početnica. Amplificirana HV-1 regija je prvo kvantificirana, a potom i sekvencirana Sangerovom metodom na automatiziranom DNA sekvenceru. Oslanjajući se na tako prikupljene podatke, kreirane su početnice specifične za odabrana žarišta, i provedena je njihova pojedinačna detekcija metodom jednobazne ekstenzije. Nakon toga, kreirane početnice su pomiješane u multipleks reakcije, koje su provedene prvo na individualnim uzorcima, te potom na miješanom uzorcima. U istraživanju provedenom u okviru ove disertacije korišteni su ABI BigDye v1.1, ABI SNAPShot i ABI Quantifiler kitovi, a strojna analiza je napravljena koristeći ABI 3130 Genetic Analyzer i ABI 7700 Sequence Detection System instrumente. ----- Rezultati: analizom elektroferograma sekvenciranja utvrđena su žarišta pojavljivanja SNP-ova, i izračunate zastupljenosti alternativnih inačica SNP-ova na njima. Dobivene sekvence HV-1 regije su podijeljene u 33 tipa mtDNA. Za sve tipove izračunate su vrijednosti GGIP (Gornje Granice Intervala Pouzdanosti), i LR (Likelihood Ratio). Rezultati analize jednobaznom ekstenzijom pokazali su se u potpunosti podudarni s rezultatima sekvenciranja po Sangeru, kako u reakcijama s pojedinačnim početnicama, tako i u multipleks reakcijama. Analize miješanih uzoraka DNA jasno su tipizirale oba tipa mtDNA prisutnih u mješavini. ----- Zaključci: na osnovu dobivenih rezultata, a u skladu s postavljenim ciljevima ovog istraživanja i njihove usporedbe s dosadašnjim istraživanjima, dolazi se do sljedećih zaključaka: izdvojeno je petnaest SNP žarišta čiji lokusi i zastupljenosti alternativne inačice SNP-a tvore jedinstvenu populacijsku studiju, što je izraženo različitim izračunatim vrijednostima statističke signifikantnosti podudaranja unutar kreirane studije naspram sličnih studija iz literature. Kreirana metoda detekcije profiliranja SNP-ova na utvrđenim žarištima jednobaznom ekstenzijom pokazala se funkcionalnom, a njeni rezultati u potpunosti podudarni s rezultatima sekvenciranja. Također, kreirana metoda detekcije jednobaznom ekstenzijom pokazala je visoku osjetljivost u tipiziranju miješanih uzoraka, području nepokrivenom metodom sekvenciranja po Sangeru.Aim of this study was to establish locations of the SNP hotspots in the HV 1 region, their alternative allele frequencies, as well as creation of a protocol for their typing based on the single base extension (SBE). ----- Materials and Methods: DNA was extracted from buccal swabs taken from 300 unrelated individuals. Extracted DNA was first quantified using RT-PCR, and then amplified at the HV-1 region of the mtDNA with adequate primers. Resulting amplicons were sequenced by Sanger sequencing using an automated DNA sequencer. Relying on the data thus acquired, primers specific for the chosen hotspots were designed, and samples were typed using SBE method for each SNP in single reactions. After that, designed primers were mixed in multiplex reaction mixes, and samples were typed with those. Created mixed samples from two different DNAs were tested with both singleplex and multiplex reactions. ----- Results: SNP hotspots and their alternative allele frequencies were determined. Taking these in consideration 33 mtDNA types were observed, with Upper Confidence Interval values and Likelihood Ratios calculated for each type. Results obtained from SBE typing protocol of the samples were identical to Sanger sequencing, both in singleplex and multiplex reactions. SBE protocol successfully typed mixed DNA samples. ----- Conclusion: Taking into consideration both the loci and the alternative allele frequencies of selected 15 SNPs, it can be concluded that they make a unique population study, which is shown with comparing calculations of the given mtDNA profile uniqueness using this study data and the data from studies found in the literature. Chosen SNP typing by the SBE protocol has shown itself functional, with results absolutely matching those acquired with Sanger sequencing. Furthermore, SBE protocol has shown high sensitivity in typing mixed DNA samples

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

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

Antibody seroprevalence against SARS-CoV-2 within the Canton of Sarajevo, Bosnia and Herzegovina—One year later

Background: Serostudies are important resources when following pandemics and predicting their further spread, as well as determining the length of protection against reinfection and vaccine development. The aim of this study was to update data on the prevalence of seropositive individuals in Canton Sarajevo, Bosnia and Herzegovina (B&H) from September 2020 to May 2021. Methods: Anti-SARS-CoV-2 antibodies were quantified using an electrochemiluminescence immunoassay. Results: Compared to the period April-July 2020, when anti-SARS-CoV-2 antibodies were detected in 3.77% of samples, one year later (May 2021) the estimated percentage within the same population of the urban Canton Sarajevo was 29.9% (5,406/18,066). Of all anti-SARS-CoV-2 Ig-positive individuals, 53.27% were men, and 69.00% were of 50 years of age or younger. Also, the current update found the individuals 50 years of age or younger to be more frequently anti-SARS-CoV-2 Ig positive compared to older individuals. On the other hand, higher median anti-SARS-CoV-2 Ig levels were found in individuals > 50 years old than in younger individuals, as well as in men compared to women. Seropositivity gradually increased from September 2020 to May 2021, with the lowest frequency of positive cases (3.5%) observed in September 2020, and the highest frequency (77.7%) in January 2021. Conclusion: Our results provided important seroprevalence data that could help in planning restrictive local public health measures to protect the population of Sarajevo Canton, especially considering that at the time of the study the vaccines were virtually inaccessible to the general population not belonging to any of the high-priority groups for vaccination