Presentation number: YIA 1


 Elena Essel1, Elena I. Zavala1, Ellen Schulz-Kornas2,5, Anna Schmidt1, Birgit Nickel1, Julia Richter1, Sarah Nagel1, Mateja Hajdinjak1,3, Marie Soressi4, Matthias Meyer1

1Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, Leipzig, Germany, 2University of Leipzig, Department of Cariology, Endodontology and Periodontology, Leipzig, Germany, 3Francis Crick Institute, London, UK, 4Faculty of Archaeology, Leiden University, Leiden, the Netherlands, 5Max Planck Institute for Evolutionary Anthropology, Department of Human Evolution, Leipzig, Germany

In contrast to skeletal remains and sediments, which are widely used sources of ancient faunal and human DNA, human-made artefacts from Palaeolithic sites are not commonly utilized for genetic investigations into the past. We hypothesized that tools and ornaments prepared from faunal bones and teeth may not only preserve DNA from the animals they belonged to, but could also trap DNA from the hominin individuals who made and/or used them. As such artefacts are often too precious for destructive sampling, we developed a non-destructive DNA extraction method that uses a temperature-controlled release of DNA by immersing artefacts in phosphate buffer. We then applied this method to a set of ten Pleistocene bones and teeth that were similar in size and shape to materials typically used for artefact production. Quantitative 3D surface texture measurements conducted before and after DNA extraction showed no substantial surface alterations, in contrast to another method previously suggested for non-destructive DNA extraction. When applied to a set of eleven artefacts from the Châtelperronian layers of the Quinçay site in France (excavated and cleaned more than 30 years ago), our method enabled the recovery of ancient mammalian mitochondrial DNA from two of the samples. DNA sequences were assigned to Cervidae and Elephantidae, in agreement with the morphological identification of the artefacts. Alas, the majority of sequences (70.9 and 98.3%) originated from recent human DNA contamination introduced during and after excavation, hampering our ability to detect traces of ancient hominin DNA that may point to the makers/users of these artefacts. In summary, we present a method for isolating DNA from ancient artefacts prepared from bones and teeth while preserving not only their visual appearance, but also their structural integrity. Moreover, we demonstrate that our method is in principle suitable for identifying the source material of artefacts in cases where morphological identification is difficult. Further investigations would require both, material excavated under cleaner conditions (e.g., using gloves) as well as limited subsequent handling to increase the probability that DNA from the makers/users of the artefacts can be recovered.

Key words: Non-destructive DNA extraction, temperature-controlled extraction, artefacts, ancient DNA

Presentation number: YIA 2


Vid Matišić1, Petar Brlek1, Vilim Molnar1, Eduard Pavelić1, Martin Čemerin2, Kristijan Vrdoljak2, Andrea Skelin1,3, Damir Erceg1,4,5,6, Davor Moravek1, Ivana Erceg Ivkošić1,6, Dragan Primorac1,6,7,8,9,10,11,12,13,14

1St. Catherine Specialty Hospital, Zagreb, Croatia, 2Medical School, University of Zagreb, Zagreb, Croatia, 3Genos Glycoscience Research Laboratory, Zagreb, Croatia, 4Srebrnjak Children’s Hospital, Zagreb, Croatia, 5Croatian Catholic University, Zagreb, Croatia, 6University of Osijek Faculty of Dental Medicine & Health, Osijek, Croatia, 7University of Split School of Medicine, Split, Croatia , 8Department of Biochemistry & Molecular Biology, The Pennsylvania State University, State College, PA, USA, 9The Henry C Lee College of Criminal Justice & Forensic Sciences, University of New Haven, West Haven, CT, USA , 10The National Forensic Sciences University, Gandhinagar, Gujarat, India, 11University of Rijeka, School of Medicine, Rijeka, Croatia, 12Medical School REGIOMED, Coburg, Germany, 13School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 14Medical School, University of Mostar, Mostar, Bosnia and Herzegovina

The field of pharmacogenomics is still in its early stages. However, multi-gene panel-based pharmacogenomic tests are readily available for both clinicians and patients. In the Republic of Croatia, single-gene testing has been available for over a decade; however, commercial panel-based tests targeting multiple genes known to influence drug response is a new concept that was implemented in 2018 at St. Catherine Hospital. This cross-sectional study aimed to report the prevalence of actionable pharmacogenetic interventions in patients who had undergone pharmacogenetic testing using the RightMed 27-gene panel. Retrospective analysis of single-center electronic health records was performed, including a total of 319 patients. Patients underwent pharmacogenomic testing by the RightMed panel using a TaqMan quantitative real-time PCR method and copy number variation (CNV) analysis to determine the SNPs in the 27 targeted genes from 2018 until 2022. Actionable drug-gene pairs were found in 235 (73.7%) patients. Relevant guidelines on genotype-based prescribing were available for 133 (56.7%) patients at the time of testing. Based on the patients’ genotype, 139 (43.6%) patients were using at least one drug with significant pharmacogenetic interactions, potentially predisposing them to adverse drug reactions or lack of therapeutic response. Two out of three patients in our practice were found to have at least one gene-drug interaction; therefore, the next step in personalized medicine is integrating pharmacogenomic data into patients’ electronic health records to optimize drug therapy.

Key words: pharmacogenomics, clinical application, adverse drug reactions

Presentation number: YIA 3


Karlo Miškec1, Anika Mijakovac1, Azra Frkatović2, Jasminka Krištić2, Ana Vujić1, Gordan Lauc2,3, Aleksandar Vojta1, Vlatka Zoldoš1

1University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia

Alternative glycosylation of immunoglobulin G (IgG) Fc region has a crucial role in defining pro- or anti-inflammatory effector function of the antibody. Gene network involved in regulation of IgG glycosylation is still poorly understood because glycoyltransferases and glycosydases are not the major players in this regulatory process. In this study we functionally validated gene loci associated with IgG glycosylation in previous genome-wide association studies (GWAS). We utilized established stably transfected cell line FreeStyle™ 293-F with CRISPR/dCas9 fusions for direct regulation of genes targeted with specific sgRNAs. This cell system was designed to secrete IgG molecules so that glycans on IgG can be analysed following gene manipulations. We manipulated 22 GWAS hits, grouped according to glycosylation traits such as galactosylation, fucosylation, sialylation and bisecting GlcNAC. Following gene expression manipulations, IgG glycosylation was analysed. Out of seven genes associated with galactosylation, only MANBA, HIVEP2, TNFRSF13B and EEF1A1 showed change of agalactosylated structures when upregulated using dSaCas9-VPR. Out of six hits associated with fucosylation, only upregulation of TBX21 and TBKBP1 showed significant changes in galactosylation, but no change was observed for fucosylated glycan structures. These results suggest that a different cell model might be preferable to validate different GWA hits, such as Lymphoblastoid Cell Line (LCL) which is known to be rich in fucose glycan structures. Out of five loci associated with bisecting GlcNac, upregulation of KDELR2 resulted in an increase in biantennary glycan structures with bisecting GlcNac. The upregulation of DERL2 and RRBP1 resulted in an increase of digalactosylated biantennary glycans. Out of four GWA hits for sialylation, only downregulation of SPPL3 led to hyperglycosylation with concomitant increase in sialylated and galactosylated structures and decrease in agalactosylated glycans, despite the fact that all genes were successfully up- or downregulated. Overall, these results have proven the functional role of several GWA hits which are not glycosyltransferases but are associated with the IgG glycosylation pathway. Ongoing research on LCL cell line might unravel the exact role of these and other GWAS hits.

Key words: CRISPR/dCas9, IgG glycosylation, FreeStyle™ 293-F Cells, GWAS

Presentation number: YIA 4


Amira Nabil1, Aurélien Trimouille2, Sahar El Shafei1, Nihal El Shakankiri3, Ahmed Habib4, Heba Morsy1, Angèle Tingaud-Sequeira2, Caroline Rooryck2

1Medical Research Institute, Alexandria University, Department of Human Genetics, Alexandria, Egypt, 2CHU de Bordeaux, Service de Génétique Médicale, Centre de Référence Anomalies du Développement et Syndromes Malformatifs, Bordeaux, France, 3Faculty of medicine, Alexandria University, Department of ophthalmology, Alexandria, Egypt, 4Faculty of dentistry, Alexandria University, Department of Maxillofacial and plastic surgery, Alexandria, Egypt

Oculoauriculovertebral spectrum (OAVS) is the second most frequent malformative disorder of head and neck with highly heterogeneous etiology and pathogenesis. Genetic causes have been brought up due to the existence of familial cases and numerous chromosomal abnormalities have been associated with this spectrum. Retinoic acid (RA) signaling pathway has been implicated in various developmental processes and is essential for craniofacial development. Interestingly, MYT1; the first described candidate gene for OAVS belongs to RA induced transcriptome. The molecular study was carried out on 32 clinically suspected OAVS Egyptian cases with no history of teratogenic insult or numerical chromosomal aberrations using array comparative genomic hybridization, next generation sequencing panel testing, whole exome sequencing and Sanger sequencing of ALX genes in three clinically suspected Oculo-auriculo-frontonasal spectrum cases. Array-CGH revealed dosage anomalies in 6 patients. NGS panel testing revealed missense variants of unknown significance in ZYG11B and HMX1 genes in 2 patients. One missense de novo heterozygous probably pathogenic variant was identified in DGKD gene through WES, performed for a selected trio. This variant was not reported in any databases and is annotated probably damaging by different bioinformatic tools. ALX genes screened were negative for deletions in all patients. Sanger confirmation, segregation analysis followed by functional studies for all variants and genes of interest is highly recommended to reveal their candidacy to OAVS. This research work might represent a strong initiative for future studies based upon the various CNVs and variants of interest detected and may set a paradigm for molecular diagnosis of cases with overlapping phenotypes.

Key words: OAVS, array CGH, NGS, WES, DGKD

Presentation number: YIA 5


Rachelle Turiello, James P. Landers

University of Virginia, Department of Chemistry, Charlottesville, Virginia, USA

For those evidence-producing criminal cases that lack genetic reference material for comparison and are ineligible for or non-producing of database matches, the human epigenome has been suggested as a reservoir of information for female sex typing, monozygotic twin individualization, body fluid identification, behavioral trait prediction, and DNA phenotyping by estimation of human chronological age. In particular, more than 300 research studies have been published suggesting the utility of methylation status at specified genetic loci for approximation of human age. However, the most commonly employed strategies for epigenetic analysis require sodium bisulfite conversion (BSC), a sample preparation step to preferentially deaminate unmethylated cytosines to uracil, leaving methylated cytosines intact and distinguishable for downstream analysis. Unfortunately, conventional BSC techniques are characterized by extensive DNA loss and require time-consuming, labor-intensive workflows with a high propensity for contamination. We propose a microfluidic solution for forensic epigenetic sample preparation that leverages centrifugal force to enable rapid, efficient conversion of forensically-relevant DNA input masses in a closed, automated microCD (µCD) format. Faster conversion rates and increased DNA recovery are possible via the enhanced surface-area-to-volume ratio specific to the microfluidic strategy. The method was designed with multiplexing in mind and assessed with methylation standards by multiple downstream analytical processes, including real-time polymerase chain reaction (RT-PCR), high resolution melting (HRM), and pyrosequencing. Early phase goals of this project included testing the chemistry at the microfluidic scale, adjusting the parameters of the reaction step most commonly associated with DNA loss, optimizing microfluidic architecture, and completing preliminary µCD BSC. Assay characterization was completed with primers targeting age-associated loci, ELOVL2 and FHL2. Our approach enabled reduction of incubation intervals, thereby decreasing the total assay time, with increased DNA recovery and comparable conversion efficiency to a gold-standard method.

Key words: Sodium Bisulfite Conversion, Forensic Epigenetics, DNA Phenotyping, Rotational Microfluidic



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.