Presentation number: CSHG 1

ACETABULAR PROTRUSION – UNDERESTIMATED BUT FREQUENT DEFORMITY IN PATIENTS WITH OSTEOGENESIS IMPERFECTA

Darko Antičević1,2, Željko Jeleč1,2, Dragan Primorac1,2,3,4,5,6,7,8,9,10

1School of Medicine, University of Osijek, Osijek, Croatia, 2St. Catherine Hospital, Zagreb, Croatia, 3School of Medicine, University of Split, Split, Croatia, 4University Department of Forensic Sciences, University of Split, Split, Croatia, 5Faculty of Dental Medicine and Health, University of Osijek, Osijek Croatia, 6University of Rijeka, School of Medicine, Rijeka, Croatia, 7Eberly College of Science, Pennsylvania State University, University Park, PA, USA, 8Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA, 9Medical School REGIOMED, Coburg, Germany, 10The National Forensic Sciences University, Gandhinagar, Gujarat, India

Acetabular protrusion (AP) is common deformity in people with osteogenesis imperfecta (OI) or brittle bone disease, a genetic disease of the connective tissues caused mainly by mutations in collagen type I. Clinical features of OI are brittle bones, low-energy fractures, skeletal deformities, joint laxity, blue sclerae, dentinogenesis imperfecta, cardiovascular and respiratory problems and hearing loss. Research on AP in OI is limited. However, AP is found in about 50% of people with OI, with even increase to 70% in people with OI type III. The purpose of this text is to draw attention to this frequently unrecognized deformity. We reviewed recently published articles in orthopaedic literature and compared data from literature with data on patients with OI treated in our hospital in the last five years (2016. – 2021.). Radiographs of hips and pelvis in our 17 patients (10 females and 7 males) were reviewed. Literature review has shown that AP is predominantly responsible for gastrointestinal problems and for increased risk for proximal femoral fractures and particularly for femoral neck fractures in OI population. We found one patient with OI type I, thirteen patients with OI type III; two patients with OI type IV and one patient with X-linked type OI. In our cohort, 65% of our patients with OI type III had AP of some degree. Three adult patients had significant AP as measured according to Kohler line. Two of them had obstruction constipation as gastro-intestinal complication of AP and this was resolved with dietary adjustments. No patients had femoral neck fracture, so far. This could be explained that majority of our patients were children and had limited ambulation prior long bone corrective surgery with expandable intra-medullary implants. There are single case reports of technical surgical problems in urologic prostate surgery due to AP in adult patient with OI type I, as well as carcinoma of left colon presenting as mechanical obstruction in a patient with osteogenesis imperfecta type III. In multidisciplinary management approach for patients with OI one should not underestimated deformity of pelvic bones, AP in particular, as there are significant potential problems that can affect quality of life in this population.

Key words: osteogenesis imperfecta, acetabular protrusion, treatment, prevention


Presentation number: CSHG 2

THE ROLE OF PHARMACOGENOMICS IN EVALUATING THE EFFICACY AND SAFETY OF DRUGS

Lidija Bach-Rojecky1, Elizabeta Topić2, Mario Štefanović3, Dragan Primorac4,5,6,7,8,9,10

1Department of Pharmacology, University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, 2Croatian Society of Medical Biochemists, Zagreb, Croatia, 3University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia, 4St. Catherine Specialty Hospital, Zagreb and Zabok, Croatia, 5 School of Medicine,University of Split, Split, Croatia, 6Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 7School of Medicine, University of Rijeka, Rijeka, Croatia, 8Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 9Eberly College of Science, State College, Penn State University, PA, USA, 10The Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA, 11Medical School REGIOMED, Coburg, Germany

To achieve a pharmacological activity, drugs should undergo passage through cell membranes from the site of application, transport to cell compartments, biotransformation to active or inactive metabolites, elimination from the body, and finally bind to specific biological macromolecules (molecular targets). The biological molecules involved are metabolic enzymes and transporters, membrane/cell receptors, and other intra- or extracellular proteins.  The polymorphisms of the genes encoding these proteins can lead to changes in the drug’s pharmacological effect, resulting in therapeutic failure and/or the occurrence of adverse drug reactions. Variations in genes involved in drug absorption, distribution, metabolism, and elimination (ADME) can alter its pharmacokinetic profile, influencing systemic exposure and concentration at the site of action. Additionally, variations associated with a drug’s target molecules can directly affect its pharmacodynamic effect. In the last twenty years, pharmacogenomics has attracted attention as a discipline that can contribute to the quality of patient health care and present an essential part of the personalized medicine concept. The use of genetic information would help predict the response to the drugs to enable safer, more effective, and cost-effective treatment to each patient.


Presentation number: CSHG 3

INFLUENCE OF GENETIC SUB-STRUCTURING OF STATISTICAL FORENSIC PARAMETERS ON GENETIC STR MARKERS IN THE POPULATIONS OF SOUTHEASTERN EUROPE

Natalija Novokmet1, Marijana Peričić Salihović1, Vedrana Škaro1,2, Petar Projić1,2, Jelena Šarac1, Dubravka Havaš Auguštin1, Saša Missoni1,3, Pavao Rudan4, Dragan Primorac3,5,6,7,8,9,10,11,12,13, Damir Marjanović1,14

1Laboratory for Molecular Anthropology, Center for Applied Bioanthropology, Institute for Anthropological Research, Zagreb, Croatia, 2DNA Laboratory, Genos Ltd., Zagreb, Croatia, 3School of Medicine, University of Osijek, Osijek, Croatia, 4Scientific Council for Anthropological Research, Croatian Academy of Sciences and Arts, Zagreb, Croatia, 5St. Catherine Hospital, Zagreb, Croatia, 6School of Medicine, University of Split, Split, Croatia, 7University Department of Forensic Sciences, University of Split, Split, Croatia, 8Faculty of Dental Medicine and Health, University of Osijek, Osijek Croatia, 9University of Rijeka, School of Medicine, Rijeka, Croatia, 10Eberly College of Science, Pennsylvania State University, University Park, PA, USA, 11Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA, 12Medical School REGIOMED, Coburg, Germany, 13The National Forensic Sciences University, Gandhinagar, Gujarat, India, 14Department of Genetics and Bioengineering, International Burch University, Sarajevo, Bosnia and Herzegovina

The goal was to perform a meta-analysis of synthesized data and investigate the influence of specific intrapopulation genetic structures on interpopulation relationships. Special focus was the influence of island population isolation on the substructuring of the Croatian population, and the influence of regional population groups on the substructuring of Southeast Europe. A long-term goal is to develop a model of appropriate sampling of the total population when creating a database of genetic STR markers that would properly reflect all the characteristics of included subpopulations. Autosomal STR loci were analyzed using four forensic parameters (match probability, power of discrimination, power of exclusion and the degree of polymorphism) on a sample of 2877 unrelated participants of both sexes. The analysis was performed using the statistical package PowerStats v1.2. The comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and certain rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub)populations should be taken into account for appropriate sampling of the total population when creating a DNA database of STR markers.

Key words: STRs, genetic sub-structuring, forensic parameters, Croatia, Southeast Europe


Presentation number: CSHG 4

EXOME SEQUENCING AND AUTISM SPECTRUM DISORDER- DIAGNOSTIC CHALLENGES AND FUTURE DIRECTIONS

Ljubica Odak, Katarina Vulin, Ana-Marija Meašić, Adriana Bobinec, Ivona Sansović,

Children’s Hospital Zagreb, Centre of Excellence for Reproductive and Regenerative Medicine, Medical School University of Zagreb, Zagreb, Croatia

The genetic basis of autism spectrum disorder (ASD) is still poorly understood in many patients. Next-generation sequencing enables simultaneous detection of pathogenic variants in hundreds of disease-causing genes. The goal of this study is to define diagnostic utility of clinical exome sequencing in elucidating autism spectrum disorder (ASD) etiology and create future diagnostic and therapeutic directions. Material and Methods For this study, we analyzed 55 ASD patients that were diagnosed and treated at the Department of Medical Genetics and Reproductive Health in Children’s Hospital Zagreb. All of them had a confirmed ASD diagnosis and were underwent to detailed clinical geneticist’s evaluation. Chromosomal disorders and fragile X syndrome have been previously excluded in all patients. Clinical exome sequencing has been performed using Illumina TruSight One Kit. In 14 out of 55 patients (25.4%) pathogenic variants were identified (14/55) and involved genes: CAMTA1, DEAF1, EP300, DICER1, MED13, CHD7, DYRK1A, FOXG1, SOS1, MED12, EHMT1, CHD8, TCF4, NFIX, PAK3. Variants of unknown significance (VUS) were present in 16.4 % of patients (9/55), in genes: AUTS2, DLG3, IHIF1, DEAF1, BICRA, CREBBP, CTNNB1, RELN. The remaining patients had negative test results (32/55; 58.2%). Clinical exome sequencing elucidated genetic basis of autism in 25 % of ASD patients, mostly attributable to genes involved in fundamental genetic processes; transcription (CAMTA1, DEAF1, EP300, MED13, FOXG1, MED12, TCF4, NFIX), chromatin remodeling (CHD7, CHD8, EHMT1), ribonuclease activity (DICER1) and activity of various kinases (DYRK1A, SOS1, PAK3). High percentage of negative and inconclusive (VUS) results (74.6%) requires additional genetic (whole exome/genome), epigenetic and environmental risk factor analysis. Establishing autism genetic basis is the prerequisite for the development of new therapeutic strategies and personalized treatment for the ASD patients. Acknowledgment: This work was supported by Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine – Exploring New Platforms and Potentials”

Key words: autism, exome sequencing, personalized management


Presentation number: CSHG 5

SEX-RELATED IMMUNOPHENOTYPE DIFFERENCES IN HUMAN STROMAL VASCULAR FRACTION FROM LIPOASPIRATE AND MICROFRAGMENTED LIPOASPIRATE REVEALED BY POLYCHROMATIC FLOW CYTOMETRY

Denis Polancec1, Lucija Zenic1, Damir Hudetz2,3,4, Zeljko Jelec2,5, Eduard Rod2, Dinko Vidovic2,6,7, Mario Staresinic8,9, Srecko Sabalic6,10, Trpimir Vrdoljak2,3, Tadija Petrovic6, Fabijan Cukelj6,10, Vilim Molnar2,4, Martin Cemerin2,9, Vid Matisic2, Petar Brlek2, Zrinka Djukic Koroljevic2, Igor Boric2,11,12,13, Gordan Lauc14,15, Dragan Primorac2,4,10,11,12,16,17,18,19,20,21

1Srebrnjak Children’s Hospital, Department for Translational Medicine, Zagreb, Croatia, 2St. Catherine Specialty Hospital, Zagreb, Croatia, 3Clinical Hospital Sveti Duh, Zagreb, Croatia, 4School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 5Department of Nursing, University North, Varaždin, Croatia, 6University Hospital Sestre Milosrdnice, Clinic for Traumatology, Zagreb, Croatia, 7School of Dental Medicine, University of Zagreb, Zagreb, Croatia, 8Department of Traumatology, Medical University Merkur Hospital, Zagreb, Croatia, 9Medical School, University of Zagreb, Zagreb, Croatia, 10Medical School, University of Split, Split, Croatia, 11Medical School, University of Rijeka, Rijeka, Croatia, 12Medical School, University of Mostar, Mostar, Bosnia and Herzegovina, 13Department of Health Studies, University of Split, Split, Croatia, 14Genos Glycoscience Research Laboratory, Zagreb, Croatia, 15Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, 16Eberly College of Science, The Pennsylvania State University, University Park, State College, PA, USA, 17The Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA, 18Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 19Medical School REGIOMED, Coburg, Germany, 20The National Forensic Sciences University, Gandhinagar, Gujarat, India, 21University of Split, University Department of Forensic Sciences, Split, Croatia

Mesenchymal stem/stromal cells or from recently referred to as medicinal signaling cells (MSC) hold tremendous therapeutic potential in regenerative medicine. Although successfully used for treating knee osteoarthritis (OA), a broader application of MSC in the field requires a better understanding of functional and cellular heterogeneity. In order to gain insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of stromal vascular fraction (SVF) from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry. Sixteen OA patients (eight females and eight males) were enrolled in the study. For each of the patient SVF was obtained from lipoaspirate and microfragmented lipoaspirate counterpart. Isolated SVF cells were stained with fluorochrome-labelled antibodies specific for the CD31, CD34, CD45, CD73, CD90, CD105, CD146 cell surface markers and analyzed by flow cytometry. We found an enrichment of the endothelial progenitor cells in the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate; in female patients it contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supraadventitial-adipose stromal cells. Some of these MSC-expression differences were present after microfragmentation, which indicates a diverse phenotype pattern of the applied MSC in female and male patients. Adding to the understanding of the perplexed heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, our results tackle on the sex-related molecular signatures in women and men that might contribute to a personalized approach of regenerative medicine.

Key words: osteoarthritis, mesenchymal stem/stromal cells, medicinal signaling cells, stromal vascular fraction, immunophenotyping


Presentation number: CSHG 6

CROATIAN GENETIC HERITAGE: RENEWED Y CHROMOSOME STORY TWO DECADES LATER

Dragan Primorac1,2,3,4,5,6,7,8,9,10, Vedrana Škaro11,12, Petar Projić11,12, Saša Missoni11, Ivana Horjan Zanki13, Siniša Merkaš13, Jelena Šarac11, Natalija Novokmet11, Andrea Ledić13, Adela Makar13, Gordan Lauc12,14, Šimun Anđelinović15, Željana Bašić3, Ivana Kružić3, Marijana Neuberg16, Martina Smolić4,5, Robert Smolić4,5, Irena Hrstić6,17, Dragan Trivanović6,17, Rijad Konjhodžić18, Lana Salihefendić18, Naida Babić Jordamović19, Damir Marjanović11,19

1St. Catherine Hospital, Zagreb, Croatia, 2University of Split, School of Medicine, Split, Croatia, 3University of Split, University Department of Forensic Sciences, Split, Croatia, 4University of Osijek, Faculty of Medicine, Osijek, Croatia, 5University of Osijek, Faculty of Dental Medicine and Health, Osijek, Croatia, 6University of Rijeka, School of Medicine, Rijeka, Croatia , 7Pennsylvania State University, Eberly College of Science, University Park, PA, USA, 8University of New Haven, Henry C. Lee College of Criminal Justice and Forensic Sciences, West Haven, CT, USA, 9Medical School REGIOMED, Coburg, Germany, 10The National Forensic Sciences University, Gandhinagar, Gujarat, India, 11Institute for Anthropological Research, Center for Applied Bioanthropology, Molecular Anthropology Laboratory, Zagreb, Croatia, 12Genos Ltd., DNA Laboratory, Zagreb, Croatia, 13Forensic Science Centre „Ivan Vučetić“, Zagreb, Croatia,14University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Pharmacology, Zagreb, Croatia, 15University Hospital Center, Split, Croatia, 16University North, Varaždin, Croatia , 17General Hospital Pula, Pula, Croatia, 18Alea Genetic Center, Sarajevo, Bosnia and Herzegovina, 19International Burch University, Department of Genetics and Bioengineering, Sarajevo, Bosnia and Herzegovina

The aim of the study was to analyze additional set of Y-Chromosome genetic markers to acquire a more detailed insight into the diversity of the Croatian population. The total number of 518 Yfiler™ Plus profiles was genotyped. Allele, haplotype frequencies and haplotype diversity, were calculated using the STRAF software package v2.0.4. Genetic distances were quantified by Rst using AMOVA online tool from the YHRD. The evolutionary history was inferred using the neighbor-joining method of phylogenetic tree construction in MEGAX software. Whit Athey’s Haplogroup Predictor v5 was used for additional comparison with selected European populations. The total of 507 haplotypes were used for genetic STR analysis. The interpopulation comparison with the original 27 Y-STR markers shows the lowest genetic diversity between Croatian and Serbian population, and the highest between Croatian and Spanish population. Interpopulation study on 17 Y-STR markers shows the lowest genetic diversity between Croatian and Bosnian-Herzegovinian population, and the highest between Croatian and Irish population. Total of 518 haplotypes were used in the determination of haplogroup diversity. Haplogroup I with its sublineage I2a expressed the highest prevalence. Haplogroup R, with its major sublineage R1a, is the second most abundant in the studied Croatian population, except for the subpopulation of Hvar, where E1b1b is the second most abundant haplogroup. Rare haplogroups also confirmed in this study are L, T and Q. G1 is detected for the very first time in Croatian population. New insight into differences between examined subpopulations of Croatia and their possible (dis)similarities with neighboring abroad populations was notified.

Key words: Y chromosome, haplogroup, Croatian population, STR, genetic heritage


Presentation number: CSHG 7

CHROMOSOMAL MICROARRAY IN CLINICAL DIAGNOSIS OF CEREBRAL PALSY

Katarina Vulin, Ljubica Odak, Leona Morožin Pohovski, Ivana Đaković, Ana Tripalo Batoš, Ana-Maria Meašić, Mijana Kero, Ivona Sansović, Adriana Bobinec, Ingeborg Barišić

Children’s Hospital Zagreb, Centre of Excellence for Reproductive and Regenerative Medicine, Medical School University of Zagreb, Zagreb, Croatia

Cerebral palsy (CP) is a group of non-progressive disorders of posture, tone, and/or movement. It is caused by a non-progressive lesion of the developing brain or brain malformation. Recent studies have implicated that genetic factors could contribute to diagnosis of CP or even cause CP. Potentially deleterious genomic copy number variants (CNVs) have been found in several CP cohorts, but estimates varied considerably depending on study criteria. The goal of this study is to define diagnostic utility of chromosomal microarray in children with CP, with well-defined phenotype according to Surveillance of cerebral palsy in Europe (SCPE) criteria. This study included 79 patients with CP, referred to the Department of Medical Genetics and Reproductive Health, Children’s Hospital Zagreb. All of them had confirmed CP diagnosis by the criteria of SCPE. The analysis was conducted using Agilent 60K oligonucleotide array-based comparative genomic hybridization. Clinically relevant variants were detected in 7 of 79 patients (8,86%): deletions 14q32.31q32.33, 1q21.1q21.2, 15q11.2, 17p13.3 and 22q11.2; duplications 1q21.1q21.2 and Xq28 (in male patient). Variants of unknown significance (VOUS) were present in 5 patients (6,33%): duplications 15q11.2, 3p26.3p26.2, 18p11.31 and Xq28 (in female patient) and deletion 17p12. Remaining patients had negative test results. Among the patients with clinically important variants, three had brain MRI classified as maldevelopment, three as predominant white matter injury, and one patient with 22q11.2 deletion as predominant grey matter injury. The genomic architecture of CP is highly complex, similar to other neurodevelopmental disorders. Continued analysis and reporting of CNV findings alongside massively parallel SNV analyses are needed to expand our knowledge of CP. Better understanding of all the possible genes involved in CP etiology is the cornerstone for understanding neurobiology of CP. This work was supported by Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine – Exploring New Platforms and Potentials”.

Key words: cerebral palsy, chromosomal microarray, copy number variants

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Published: June 21st, 2022;

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