Presentation number: PP1-MG
MiR-182-5p AND miR-375-3p IN BLOOD PLASMA AS BIOMARKERS FOR PROSTATE CANCER
Irena Abramović1,2,3, Borna Vrhovec4, Ivan Pezelj4, Tomislav Kuliš2,3,5, Alen Vrtarić6, Monika Ulamec2,3,7,8, Nino Sinčić1,2,3
1School of Medicine, University of Zagreb, Department of Medical Biology, Zagreb, Croatia, 2Scientific Group for Research on Epigenetic Biomarkers, School of Medicine, University of Zagreb, Zagreb, Croatia, 3Scientific Centre of Excellence for Reproductive and Regenerative Medicine, School of Medicine, University of Zagreb, Zagreb, Croatia, 4University Clinical Hospital Centre “Sestre Milosrdnice”, Department of Urology, Zagreb, Croatia, 5University Hospital Centre Zagreb, Department of Urology, Zagreb, Croatia, 6University Clinical Hospital Centre “Sestre Milosrdnice“, Department of Clinical Chemistry, Zagreb, Croatia, 7University Clinical Hospital Centre “Sestre Milosrdnice”, Ljudevit Jurak Clinical Department of Pathology and Cytology, Zagreb, Croatia, 8School of Medicine, University of Zagreb, Department of Pathology, Zagreb, Croatia
With prostate cancer (PCa) being the most commonly diagnosed neoplasm among men and often resembling benign prostate hyperplasia (BPH), biomarkers with a higher differential value than PSA are required. We investigated the expression of certain miRNAs from liquid biopsies as potential epigenetic biomarkers of PCa. The absolute expression of miR-375-3p and miR-182-5p were quantified in blood plasma and seminal plasma of 65 PCa and 58 BPH patients by digital droplet PCR. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. The higher expression of miR-182-5p and miR-375-3p in the blood plasma of PCa patients was statistically significant as compared to BPH (p = 0.0363 and 0.0226, respectively). Their combination achieved a specificity of 90.2 % for predicting positive or negative biopsy results, while PSA cut-off of 4 µg/L performed with only 1.7 % specificity. In seminal plasma, miR-375-3p, and miR-182-5p showed a statistically significantly higher expression in PCa patients with PSA >10 µg/L compared to ones with PSA >10 µg/L. MiR-182-5p and miR-375-3p in blood plasma show higher performance than PSA in differentiating PCa from BPH. Seminal plasma requires further investigation as it represents an obvious source for PCa biomarker identification.
Key words: miRNA, prostate cancer, liquid biopsy, biomarkers
Presentation number: PP2-FG
EXPANDED CODIS STR ALLELE FREQUENCIES – EVIDENCE FOR THE IRRELEVANCE OF RACE-BASED DNA DATABASES
Robert F Oldt, Sree Kanthaswamy
Arizona State University, AZ, USA
In the US, it is assumed that the most conservative random match probability (RMP) can be estimated using the suspect’s self-declared racial reference DNA database. If the suspect’s race is not known, the RMP is typically estimated across different race-based CODIS STR allele frequency databases, including African American, Asian, Caucasian, Hispanic, and Native American, and the most conservative RMP estimate is used. In a recent study, we evaluated the relationship between RMP and race based on CODIS STR profiles corresponding to the five race-specific allele frequency databases. Our analyses confirmed that most genetic differences between individuals are only to the slightest extent attributable to racial classification. Approximately 98% of the genetic variation was found to occur among individuals and not between races. We could not distinguish individuals separated by race as distinct genetic clusters based on forensic STR data. Accordingly, RMP values were exceedingly small regardless of the race-based STR allele frequency database, and the values also did not vary significantly when incorporating race-specific reference data. Therefore, our results show that the use of racial information does little to generate conservative RMP estimates. This finding implies that we do not need to include racial information in the US to get conservative estimates of RMP. Therefore, using race as a proxy for a genetic distinction to produce larger (i.e., conservative) RMPs for an individual DNA profile is irrelevant.
Key words: Race, random match probability, population structure, forensic DNA database
Presentation number: PP3-MG
ENRICHMENT OF RARE VARIANTS IN GENES INVOLVED IN MITOCHONDRIAL METABOLISM IN PATIENTS WITH EARLY-ONSET OR FAMILIAL PARKINSON’S DISEASE
Dorian Laslo1, Gaber Bergant2, Aleš Maver2, Borut Peterlin2
1Josip Juraj Strossmayer University of Osijek, Faculty of Medicine Osijek, Osijek, Croatia, 2Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia
There is a growing body of evidence supporting mitochondrial dysfunction as a mayor driving force in Parkinson’s disease (PD) pathogenesis, with the hallmark being the discovery of mitochondrial toxins inducing PD. The goal of this study was to analyze rare variants of genes involved in mitochondrial metabolism in patients with early-onset and familial PD. We collected both nuclear and mitochondrial variants for 204 Croatian, Serbian and Slovenian patients with PD who referred to our center for diagnostic whole exome sequencing. The 204 patients with PD were further selected based on the disease age of onset and their family history. Sequencing was performed using a standardized set of protocols. We used population variation resources in variant annotation, which included an in-housed background population variant frequency estimates based on compilation of over 7000 exomes, as well as Genome Aggregation Database dataset. The cutoff frequency for rare variants was 5% in both the local and gnomAD databases. We excluded variants deviating significantly from Hardy-Weinberg equilibrium. In our study we found statistically significant differences in 16 gene variants between PD patients and the control group. After analysis genes GBA, NPC1, ATP13A2, HTRA2, CP, SOD1, WARS2, CLN6, HEXA, NPC2, SYNJ1, WDR45, PANK2, POLG which are located in nucleus and MT-CO1 gene located in mitochondrial genome had p<0.05. After adjustment only nuclear genes still had p<0.05, while MT-CO1 gene had padj=0,381. All identified genes have important role in lysosomal or mitochondrial function. We confirmed previous findings that dysfunction in lysosomal metabolism plays an important role in pathogenesis of PD. We found that ATP13A2 mutation is more abundant in patients with early onset PD, not only juvenile form of PD. Mutation in HtrA2 gene results in protein accumulation and destabilization of mitochondrial membrane. WARS2 encodes tRNA synthetize and our study is the first one that links its mutation with early-onset PD. Our study is the first one that identified new gene, WARS2, as a risk factor for early-onset PD and confirmed that dysfunction of lysosomal or mitochondrial metabolism is risk factor for developing PD.
Key words: Lysosomes, Mitochondria, Parkinson Disease
Presentation number: PP4-FG
DEVELOPMENT AND VALIDATION OF A NEW MULTIPLEX METHYLATION-SPECIFIC PCR ASSAY FOR FORENSIC BODY FLUID IDENTIFICATION
Sasho Risteski1,2, Zlatko Jakovski1, Lozenka Ivanova2, Sasho Panov2, Gordana Dimeska2, Hilda Jovanovikj1, Ksenija Nikolova1, Aleksandar Stankov1
1Ss. Cyril and Methodius University in Skopje, Faculty of Medicine, Insitute of Forensic Medicine, Criminalistics and Medical Deontology, Skopje, Macedonia, 2Ss. Cyril and Methodius University in Skopje, Faculty of Natural Sciences and Mathematics, Institute of Biology, Skopje, Macedonia
The identification of the body-fluid from which DNA originates, along with the STR profiling, is important in uncovering the context of deposition of biological traces and can aid in reconstruction of events. Several authors have reported MS-SNuPe assays for detecting differentially-methylated CpGs for differentiation between saliva, semen, vaginal fluid, venous and menstrual blood. The aim of our research was to develop and validate a novel multiplex methylation-specific PCR (F-MS-PCR) assay for detection of methylation in 5 body-fluid specific CpGs in a DNA sample. The test consists of PCR amplification of bisulfite-converted DNA from a sample by using 5 pairs of fluid-specific primers, one of which is fluorescently tagged at its 5′-end and specific to bisulfite-converted DNA, and the other is specific to bisulfite-converted but only methylated DNA. Thus, only amplification of the methylated body-fluid specific locus is achieved, the PCR product is detected by CE and the body-fluid is detected. To assess the performance, 27 semen, 21 saliva, 21 venous blood, 21 menstrual and 20 vaginal samples were tested. DNA was extracted by PrepFiler Forensic DNA Extraction Kit, quantified by Quantifiler Duo on 7500 Real-Time PCR System, and bisulfite-converted by using EpiJET Bisulfite Conversion Kit. PCR reactions consisted of: 5 µl Qiagen Multiplex PCR Mix, 1 µl 10x primer mix, 3 µl water and 1 µl bisulfite-converted DNA. PCR products were detected on 3500 Genetic Analyzer and data was analyzed by GeneMapper ID-X software. Validation of the limit of detection, sensitivity, specificity, application in casework, and analysis of old samples and mixtures, was also performed to assess the suitability of the test for routine use in the forensic genetic laboratory. The test showed high specificity (100%, except 96.73% in menstrual fluid) and high sensitivity (100%, except 97.6% in vaginal and 80.7% in menstrual fluid). The lower limit of detection varied between 125 pg (semen) and 1 ng (menstrual blood). It has fewer pipetting steps compared to MS-SNuPe, shorter sample to result time and lower cost. The test has easy interpretation, with presence of a peak indicating detected fluid, and it can be easily applied as a powerful tool in the forensic genetic laboratory.
Key words: Body-fluids, DNA methylation, multiplex PCR, MS-SNuPe, capillary electrophoresis
Presentation number: PP5-MG
THE EFFECT OF HOST GENETICS ON COVID-19 SUSCEPTIBILITY AND SEVERITY: A STUDY OF 16 CODING GENES ON A SUBSET OF BOSNIAN-HERZEGOVINIAN PATIENTS
Lana Salihefendić1,2, Larisa Bešić1, Ivana Čeko1,2, Naida Mulahuseinović2, Dino Pećar2, Amra Džuho1, Selma Durgut2, Damir Marjanović1,3, Rijad Konjhodžić2, Adna Ašić1
1International Burch University, Department of Genetics and Bioengineering, Sarajevo, Bosnia and Herzegovina, 2Alea Genetic Center, Sarajevo, Bosnia and Herzegovina, 3Institute for Anthropological Research, Zagreb, Croatia
The aim of this study was to analyze the effect of patient genetics on the severity of symptoms and susceptibility to COVID-19 infection. 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herezgovina, were recruited in the study, and divided into three groups (n=20 for each group) of patients exhibiting mild, moderate and severe clinical presentation of COVID-19. DNA was isolated from whole blood using QIAamp® DNA Mini Kit. Ion Torrent GeneStudio S 5 platform was used to perform the sequencing of 16 target genes and their regulatory regions, namely 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. Selected genetic variants of interest were subjected to confirmatory Sanger sequencing on the SeqStudio Genetic Analyzer System. Our study confirmed that older age, male sex, and cardiovascular, respiratory and metabolic comorbidities are risk factors for severe COVID-19. In addition, we have identified several genetic variants that were significantly more common in severe than in mild and/or moderate groups of patients, including those on the genes IL4, IL1B, TMPRSS2, CD55, DDX58 and IRF7. We have further confirmed that the variant rs2285666 on ACE2 might be protective, as it was more common in moderate and mild than in severe clinical group, but significantly affected by the patient’s sex, considering that ACE2 is found on X chromosome and escapes X inactivation in females. The results of this study have further emphasized the importance of personalized approach to each COVID-19 patient, as host genetics plays an important role in response to SARS-CoV-2 infection, including both susceptibility and severity of the clinical presentation. Future studies, most prominently GWAS using larger patient cohorts and appropriately matched controls, are expected to produce even more data on the effect of human genetic variants on the course of COVID-19. This study is offering the first such data, not only for the Bosnian-Herzegovinian population, but for the Western Balkan region as well.
Key words: COVID-19, host genetics, personalized approach, SARS-CoV-2
Presentation number: PP6-MG
MOLECULAR AUTOPSY: IDENTIFICATION, CLASSIFICATION AND REPORTING OF SEQUENCE VARIATIONS IN YOUNG SUDDEN UNEXPECTED DEATH VICTIMS AND AFFECTED FAMILIES
Stefanie Scheiper-Welling1, Britt-Maria Beckmann1, Tina Jenewein2, Elise Gradhand3, Alexandra Tschäbunin3, Christof Geisen3, Marcel A. Verhoff1, Silke Kauferstein2
1University Hospital Frankfurt, Goethe University Frankfurt, Insitute of legal medicine, Frankfurt, Germany, 2University Hospital Frankfurt, German Red Cross, Institute of Transfusion Medicine and Immunohaematology, Frankfurt, Germany, 3University Hospital Frankfurt, Goethe University Frankfurt, Dr. Senckenbergisches Institute of Pathology, Frankfurt, Germany
Sudden cardiac death (SCD) is an important public health issue. In young individuals a significant number of SCDs is caused by inherited cardiac diseases, frequently not detectable during conventional medico-legal autopsies (including histological and toxicological analyses). Therefore, these deaths are referred to as sudden unexplained deaths (SUDs). Next-generation sequencing (NGS) became an indispensable tool in molecular autopsy investigations. Nonetheless, NGS brought new challenges, especially regarding the interpretation of the large number of variants of unknown significance (VUS). Therefore, evaluation and classification of genetic findings is of great importance to establish a causal link between phenotype and genotype and for prevention of the remaining family members. We performed a structural assessment of young sudden death cases aged between 1-50 years including molecular autopsy. Detected sequence variants were assessed according to the ACMG classification standards. In addition, cardiological data of our new centre of sudden death in the young were investigated to evaluate possible genotype-phenotype correlations. We identified 53 rare protein-altering variants (MAF < 0.2%) classified as VUS or worse. 13 % of the cases exhibited a clinically actionable variant (pathogenic, likely pathogenic or VUS – potentially pathogenic) that would warrant cascade genetic screening in relatives. To date, using molecular autopsy in combination with the assessment of family members (n=149), an inherited cardiac disease could be detected in 24 % of the cases. 67 % of the families are still under investigations. Our data reveal that, despite the undeniable advantages, molecular autopsy is not a stand-alone tool. Moreover, multidisciplinary collaboration is crucial for an optimal management of sudden unexplained death cases in order to identify additional relatives at risk.
Key words: Sudden death, Molecular Autopsy, inherited cardiac disease
Presentation number: PP7-MG
CELL LINE MODELS WITH STABLY INTEGRATED CRISPR/DCAS9 FUSIONS FOR STUDYING THE EPIGENETICS OF IgG GLYCOSYLATION
Aleksandar Vojta1, Anika Mijakovac1, Karlo Miškec1, Jasminka Krištić2, Vedrana Vičić Bočkor1, Gordan Lauc2, Vlatka Zoldoš1
1Department of Biology, Division of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia
To study alternative glycosylation of immunoglobulin G (IgG), which is functionally important in in pro- or anti-inflammatory effector function of the antibody, we developed a system for IgG production in the model cell line HEK-293F, which we modified by incorporating the CRISPR/dCas9-based molecular machinery for epigenetic regulation of gene expression. We extended our existing modular system for CRISPR/dCas9 fusions facilitating gene regulation to enable its stable integration into HEK-293F cells, thus creating derived cell lines with integrated machinery for programmed transcriptional control. A key factor for targeting the dCas9 fusions with activators and repressors, the guide RNA (gRNA) was transiently transfected into the derived cell lines on a separate plasmid, that also contains a cassette for monocistronic expression of IgG heavy and light chain. In this bipartite system, the creation of derived cell lines with CRISPR/dCas9 components eliminates the inefficiency of transfection with a large construct, while combining a key element for targeting (gRNA) with the IgG expression cassette on a small plasmid facilitates high transient transfection efficiency while virtually eliminating the background from untransfected cells. We validated the system by up- or downregulating the known glycosyltransferases. Transcriptional up- and downregulation of B4GALT1, responsible for adding galactose to the glycan core, had the expected effect on the abundance of agalactosylated and galactosylated structures, changing the glycosylation profile according to the transcript level. Downregulation of FUT8, encoding a fucosyltransferase, decreased the core-fucosylated structures, while upregulation of ST6GAL1 and MGAT3 increased sialylated structures and those with a bisecting GlcNAc, respectively, in line with our expectations. The system is now being used to further study the role of genes associated with IgG glycosylation in GWA studies. It can also be easily repurposed to serve as a model for other proteins and their posttranslational modifications, with appropriate targeting via gRNA. Finally, to eliminate the frequently raised concern about suitability of the HEK293-F system as a model for plasma cells, we are currently adapting the system for use with lymphoblastoid cell lines (LCLs).
Key words: epigenetics, glycosylation, CRISPR, Cas9
Presentation number: PP8-AG
WEST COUNTRY STORY – A DETAILED INVESTIGATION OF NEOLITHIC & BRONZE AGE INDIVIDUALS FROM SOUTH-WESTERN ENGLAND
Nikola Vukovic
Uppsala University, Evolutionsbiologiskt Centrum EBC, Uppsala, Sweden
Six thousand years before present (BP), the Neolithic expansion to Britain displaced local hunter-gatherer societies, introducing a radically different, more sedentary lifestyle. Two millennia later, a new migratory wave reached Britain, through the Bell Beaker folk, again transforming the established genetic and socio-cultural landscape of the island. While distinct burial rituals and material artifacts underline the strong cultural differences between the two populations, the Bronze Age practice of reusing more ancient Neolithic tombs renders the differentiation of individuals through archaeological study alone problematic. In order to finely investigate the population dynamics that occurred during the transition between the Neolithic and the Bronze Age in Britain at an individual resolution, we employed a genomic approach on 30 samples (age between 6000-3500 BP) from 7 sites in South-Western England. Our analyses reveal a pattern consistent with a genetic turnover, while signs of admixture between the populations are visible as well. First kin relationships between four individuals provide a direct insight into the makeup of a Neolithic multigenerational family. On three occasions, we find the remains of male Bronze Age individuals buried within Neolithic sites, suggesting a possible trend of burial site reuse. By integrating genetic and archaeological analyses, our study delivers a snapshot of one of the most influential demographic shifts in British history, shedding light on both the individual history of these tombs as well as the region’s wider socio-cultural transformation.
Key words: Neolithic, Bronze Age, Migration
Published: June 21st, 2022;
Copyright: © 2022 ISABS & IAR Publishing. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
