Presentation number: MG 8

CASE REPORT: A MULTIDISCIPLINARY APPROACH TO THE MANAGEMENT OF RICKETS DISEASE CAUSED BY DE NOVO MUTATION IN THE PHEX GENE

Eduard Pavelić1, Petar Brlek1, Vilim Molnar1, Darko Antičević1,2, Damir Hudetz1, Vide Bilić1,3, Ivo Barić4, Dragan Primorac1,2,5,6,7,8,9,10,11,12

1St. Catherine Specialty Hospital, Zagreb, Croatia, 2Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 3Clinical Hospital of Traumatology, Sestre milosrdnice University Hospital Center, Zagreb, Croatia, 4Department of Paediatrics, University Hospital Center Zagreb and University of Zagreb School of Medicine, Zagreb, Croatia, 5Medical School, University of Rijeka, Rijeka, Croatia, 6Medical School, University of Mostar, Mostar, Bosnia and Herzegovina, 7Medical School, University of Split, Split, Croatia, 8Department of Biochemistry & Molecular Biology, The Pennsylvania State University, State College, PA, USA, 9The Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA, 10Medical School REGIOMED, Coburg, Germany, 11School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 12National Forensic Sciences University, Gujarat, India

Rickets disease has been a persistent disease, first described in the Viking populations throughout the centuries. However, the cause of this form of primary Rickets was the lack of vitamin D. There are secondary forms of Rickets in which lie a genetic component. X-linked hypophosphatemia (XLH) is a disorder of renal phosphate wasting caused by pathogenic variants in the phosphate-regulating endopeptidase gene, PHEX – essential to the phosphate homeostasis in the body. Biochemical findings include hypophosphatemia with low to normal circulating 1,25-dihydroxy vitamin D levels, elevated serum alkaline phosphatase activity in children, and normal serum calcium. XLH demonstrates variable expressivity, even within families, and has complete penetrance in both males and females. Symptoms can range from extreme lower limb bowing apparent by the first year of life to isolated short stature in otherwise asymptomatic appearing adults. We present a case of a 30-year-old female patient diagnosed with Rickets disease at the age of 3, which reacted unsuccessfully to treatment with vitamin D. The patient presented to our clinic for orthopedics at St. Catherine Specialty Hospital in 2021 with severe scoliosis and varus deformity of the lower extremities. Upon discussion, the patient’s anamnesis revealed a history of hypophosphatemia. Genetic testing was undergone and revealed a mutation in the PHEX gene (c.1646-1G>C) confirming the diagnosis of x-linked hypophosphatemia. The pathogenic variant (c.1646-1G>C) was not previously present in population databases, but algorithms that predict the effect of sequence changes on RNA splicing suggested this novel mutation disrupts the acceptor splice site in intron 15 of the PHEX gene and lead to a loss of protein function. After an extensive conversation with the patient, the decision for operative management was taken. Laboratory workup showed decreased phosphate levels at 0.66 mmol/L and hemoglobin at 85 g/L, while calcium and parathyroid hormone were increased at 2.58 mmol/L and 9.02 pmol/L, respectively. Substitution therapy led to the normalization of laboratory findings. After detailed medical examination, surgery in the form of a corrective osteotomy was agreed upon, which will improve the patient’s quality of life.

Key words: Rickets disease, PHEX mutation, hypophosphatemia, RNA splicing


Presentation number: MG 9

CCR5-Δ32 GENE VARIANT IN CELIAC DISEASE

Nada Starčević Čizmarević1, Vanja Licul2, Brankica Mijandrušić Sinčić2, Anika Lorencin Pucić1, Smiljana Ristić1

1Faculty of Medicine, Department of Medical Biology and Genetics, Rijeka, Croatia, 2University Hospital Centre Rijeka, Department of Gastroenerology, Rijeka, Croatia

Celiac disease is a chronic immune-mediated enteropathy of the small intestine that is triggered in genetically susceptible individuals by the consumption of gluten in the diet. Although HLA class II genes are undoubtedly involved in the development of celiac disease, only 40% of the genetic susceptibility to celiac disease can be attributed to them. This suggests that other genes within or outside the HLA region play a role in the pathogenesis of the disease. Chemokines and their receptors are involved in numerous aspects of the immune system and have been studied in various autoimmune diseases. Deletion of 32 bp in the C-C chemokine receptor type 5 (CCR5) gene results in loss of expression of the receptor. The CCR5-Δ32 mutation has already been recognized as a modifying pathogenetic factor in type I diabetes. The inflammatory diseases type I diabetes and celiac disease co-segregate in a population, suggesting a common genetic origin. Therefore, the aim of our study was to determine the possible influence of the CCR5-Δ32 mutation on the predisposition and clinical expression of celiac disease. The study included 175 patients diagnosed with celiac disease according to the revised ESPGHAN criteria and 175 healthy controls matched for age, sex, and place of residence. Polymerase chain reaction was used to genotype the CCR5-Δ32 mutation. The allele frequency of the CCR5-Δ32 mutation was significantly higher (p=0.028) in patients than in controls. However, the effects of the CCR5-Δ32 mutation on the clinical expression of the disease did not show significant effects (p>0.05). The study showed no statistically significant differences in clinical presentation of the disease according to sex. The presence of the CCR5-Δ32 mutation influences the predisposition to celiac disease in our patients, but further studies with a larger number of patients are needed.

Key words: CCR5-Δ32, gene variant, celiac disease


Presentation number: MG 10

ASSESSMENT OF GENETIC TESTING FOR GILBERT’S SYNDROME IN BOSNIA AND HERZEGOVINA IN THE PERIOD 2014-2022

Nikolina Tomić, Naida Lojo-Kadrić, Belmina Šarić, Jasmin Ramić, Lejla Pojskić

University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

Gilbert’s syndrome is a genetic, autosomal-recessive liver disorder. It is characterized by periods of elevated levels of unconjugated bilirubin in the blood (hyperbilirubinemia). Hyperbilirubinemia is caused by reduced activity of the enzyme glucuronyltransferase (coded by UGT1A1 gene) which conjugates bilirubin making it soluble in water. Patients with Gilbert’s syndrome have 7 TA repeats on both alleles of the UGT1A1 gene (TA7 / TA7) while healthy individuals are homozygous for the TA6 allele (TA6 / TA6). Heterozygotes in most cases do not develop hyperbilirubinemia. Routine genetic testing of Gilbert’s syndrome consists of DNA isolation from the blood, PCR amplification and fragment detection on genetic analyzer. From 2014 to the beginning of 2022, 21 people were referred for Gilbert’s syndrome genetic testing in our institution. Only one patient was homozygous for the TA6 allele and one was heterozygous, the rest of them were TA7 / TA7 homozygous meaning that the diagnosis was confirmed. The most of them were between 13 and 19 years old, confirming that clinical manifestations usually occur during puberty when the concentration of steroid hormones increases the bilirubin in the blood what indicates referral to genetic testing for Gilbert’s syndrome. Also, 12 out of 21 patients were male what could support the fact that Gilbert’s syndrome is more common in men than in women. Furthermore, concerning testing for this disease in Bosnia and Herzegovina, number of tested samples per year is growing in only one institution where before the end of the first trimester of 2022, five patients were tested and in previous 7 years 1-4 samples were tested per year. This short overview shows that recognition and consciousness about Gilbert’s syndrome is growing what is very important because Gilbert’s syndrome is a benign disease that does not require specific treatment and must be distinguished from other disorders of unconjugated hyperbilirubinemia.

Key words: Hyperbilirubinemia, Gilbert’s Syndrome, genetic testing, UGT1A1 gene


Presentation number: MG 11

MICRO- AND MACRONUTRIENT PROFILE IN DOWN SYNDROME CHILDREN AND ADOLESCENTS: SYSTEMATIC REVIEW AND META-ANALYSIS

Anita Barišić1, Maja Ergović Ravančić2, Dijana Majstorović3, Jadranka Vraneković1

1Faculty of Medicine, Department of Medical Biology And Genetics, Rijeka, Croatia, 2Polytechnic in Požega, Department of Agriculture, Study of Food Technology, Požega, Croatia, 3Juraj Dobrila University of Pula, Pula, Croatia

Down syndrome (DS), an expression of complete or partial trisomy of chromosome 21, is the most common genetic disorder known to date. Nowadays, there is a great deal of clinical interest in the question of whether children with DS benefit from nutritional supplementation to improve their development, cognitive decline, and overall health, especially if started early in childhood. To date, the relevant scientific literature has not been systematically reviewed and organized. Therefore, our aim was to provide a systematic review and meta-analysis of the micro-and macronutrient profile in DS children and adolescents. This study was conducted in accordance with PRISMA guidelines. We identified all relevant case-control studies published by January 1, 2021, by searching the PubMed and Scopus databases for original English-language articles analyzing the micro-and macronutrient profile of individuals with DS. After a comprehensive analysis, 40 studies were included in the qualitative synthesis and 31 in the quantitative synthesis (meta-analysis). MetaAnalysis software, version 3.0 (Biostat, Inc., Englewood, NJ, USA) was employed for the meta-analysis. Significant results (p≤0.05) were obtained for zinc, selenium, copper, vitamin B12, sodium and calcium. Serum, plasma, and whole blood analyses showed lower zinc levels in DS compared with controls (SMD serum[95%CI]=-2.32[-3.22,-1.41]; SMD plasma[95%CI]=-1.29[-2.26,-0.31]; SMD blood[95%CI]=-1.59[-2.29,-0.89]). Similarly, plasma and blood selenium concentrations were significantly lower in DS (SMD plasma [95%CI]=-1.39[-2.26,-0.51]; SMD blood[95%CI]=-1.86[-2.59,-1.13]. Intraerythrocyte copper and serum B12 were higher in DS (SMD Cu [95%CI]=3.33[2.19,4.46]; SMD B12[95%CI]=0.89[0.01,1.77). Finally, salivary sodium and calcium were slightly elevated (SMD Na [95%CI]=1.06[0.29,1.82]; SMD Ca[95%CI]=0.49[0.16,0.83], whereas blood calcium was lower in DS children/adolescents compared to controls (SMD Ca[95%CI]=-0.77[-1.34,-0.21]). This study provides the first field overview of micro-and macronutrient profiles in DS children and adolescents. Additionally, the evidence-based foundation for future dietary interventions has also been established.

Key words: adolescents, Down syndrome, children, macronutrients, micronutrients


Presentation number: MG 12

OPITZ-KAVEGGIA (FGS1) SYNDROME AND XYY CHROMOSOMOPATHY – IS IT VARIANTS OF UNCERTAIN SIGNIFICANCE (VOUS) REALY VOUS – CASE REPORT

Nora Pušeljić1, Ema Poznić1, Silvija Pušeljić1,2, Višnja Tomac1,2, Martina Kos1,2, Ivana Serdarušić1, Paula Ivanšić1, Luka Perić3, Nika Pušeljić2

1Department of Pediatrics, Osijek University Hospital, Osijek, Croatia, 2Faculty of Medicine, University of Osijek, Osijek, Croatia, 3Department of Oncology, Osijek University Hospital, Osijek, Croatia

Opitz-Kaveggia syndrome (FGS1) (OMIM # 305450) is a very rare disorder, the prevalence is unknown, although several hundred cases have been reported. Syndrom caused by mutation in the MED12 gene located at position Xq13 recessive pattern condition that affects many parts of the body. The physical features include hypotonia, facial appearance including small, underdeveloped ears; hypertelorism, macrocephaly prominent forehead; down-slanting palpebral fissures, brain anomaly, seizures and heart defects. We report 4 months old boy, born in a second pregnancy (first pregnancy misscariage) of nonconsanguineous parents, with intrauterine growth retardation, premature birth at 32 weeks of gestation, birth weight 1.3 kg. Noninvasive prenatal testing is normal. Dysmorphic features present from birth macrocephaly, depressed nasal bridge, hypertelorism, gothic palate, microretrognathia, low-set ears, mild contractures of both knees, micropenis. Neonatal period complicated with more comorbidities – respiratory distress syndrome, prolonged mechanical ventilation, bronchopulmonary dysplasia, recurrent infections, thrombotic incidents, heart failure with include multiple muscular ventricular septal defects, atrial septal defect, agenesis of the corpus callosum and bilateral hearing impairment. Karyotyping and chromosomal microarray diagnose sex chromosome aneuploidy XYY sindrom which does not explain such a complex disease. VOUS in the MED12 gene c.3692-7A> G in hemizygous status was identified by whole exome sequencing. Parental testing shows the same variant in the mother. Opitz-Kaveggia syndrome (FGS1) is a very rare X-linked recessive disorder, caused by mutation in the MED12 gene. The MED12 gene provides instructions for making a protein that helps regulate gene activity, and MED12 protein forms part of a large complex that turns genes on and off. The MED12 protein is thought to play an essential role in development both before and after birth. Although the mutations alter the structure of the MED12 protein, it is unclear how they lead to intellectual disability, and the physical features associated with this condition. We think that this is a new pathogenic variant of the MED12 gene because the patient has all the described comorbidities of FSG1.

Key words: Opitz-Kaveggia syndrome, FGS1, XYY chromoosomopathy


Presentation number: MG 13

IL-6 AND IL-8 GENE EXPRESSION IN MALE CHILDREN WITH OBESITY: A POSSIBLY EARLY ATHEROSCLEROTIC INFLAMMATORY PREDICTOR

Robert Mujkić1, Darija Šnajder Mujkić2, Petar Šušnjara3, Anita Matić4, Karla Rožac1, Ivana Ilić1, Anđela Grgić1, Dalibor Divković5, Kristina Selthofer Relatić6

1Department of Anatomy, Histology, Embryology, Pathological Anatomy and Pathological Histology, Faculty of Dental Medicine and Health, University of J. J. Strossmayer Osijek, Croatia, 2Department of Anatomy and Neuroscience, Faculty of Medicine, University of J. J. Strossmayer Osijek, Croatia, 3Department of Physiology and Immunology, Faculty of Medicine, University of J. J. Strossmayer Osijek, Croatia, 4Department of Pathophysiology, Physiology and Immunology, Faculty of Dental Medicine and Health, University of J. J. Strossmayer Osijek, Croatia, 5Department of Surgery, Urology, Orthopedics and Physical and Rehabilitation Medicine, Faculty of Medicine, University of J. J. Strossmayer Osijek, Croatia, 6Department of Hearth and Vessel Disease, University Hospital Centre Osijek, Croatia

Atherosclerosis is one well know complication that can occur during obesity and it might have inflammation processes as important part of its pathology. In some previous study results show that interleukin 8 (IL-8) and interleukin 6 (IL-6) are associated with numerous different inflammatory processes in obese state and contribute to pathogenesis of atherosclerosis and other cardiovascular diseases (CVD). Aim of this study was to analyze if there is a correlation between IL-6 and IL-8 gene expression and other anthropometric and biochemical parameters in subcutaneous adipose tissue (SAT) of healthy male children as an early sign of metabolic dysregulation that can be connected with obesity in early childhood and have impact on later development of atherosclerosis. We determined changes in gene expression of IL-6 and IL-8 in SAT in lean and overweight/obese healthy male children. Tissue samples from SAT fat depots were obtained during surgery from 32 normal weight male children age 5,12±3,21 years, and 22 overweight/obese male children age 5,95±3,24 years, who underwent elective abdominal surgery having hernia repairs or orchidopexies at the Department for Pediatric Surgery of the University Hospital Osijek. Subject were divided in two groups by their BMI Z-score. On SAT samples immunohistochemistry for detection of CD163+ cells was performed and gene expression of IL-6 and IL-8 by quantitative RT-PCR with reference genes for data normalization was measured. Children in the overweight/obese group showed a higher expression of IL-6 (p=0,019) and IL-8 (p<0,001) in subcutaneous adipose tissue compared to normal weight children. The expression of IL-6 in SAT correlated positively with the number of CD163+ cells in same adipose tissue compartment, as has the expression of IL-8. The expression of IL-8 in SAT also correlated positively with BMI Z-score, triglyceride serum concentration, average surface area of SAT adipocytes and gene expression of COL6a3. Increased gene expression of IL-6 and IL-8 in subcutaneous adipose tissue of male children could indicate a possibility that obesity and chronic low-grade inflammatory processes in subcutaneous adipose tissue during early childhood can contribute to pathogenesis of atherosclerosis in adulthood.

Key words: gene expressions, obesity, male children, atherosclerosis


Presentation number: MG 14

CELL-LINE MODEL UNCOVERS ACTIVITY OF RUNX1 AS A POTENTIAL MODIFIER OF IMMUNOGLOBULIN G GLYCOSYLATION

Ana Cindrić1, Dražen Petrović1, Aoife Murray2, Ivan Alić2, Helena Deriš1, Dean Nižetić2, Gordan Lauc3, Jasminka Krištić1

1Genos d.o.o., Zagreb, Croatia, 2The Blizard Institute, Barts & The London School of Medicine, Queen Mary University of London, London, UK, 3Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

Down syndrome (DS) is a condition caused by trisomy 21 that entails numerous symptoms, one of which are premature signs of aging. As revealed by our recent research, these signs of premature aging are also reflected in plasma-derived immunoglobulin G (IgG) glycosylation, which is a well-known marker of biological age (Krištić et al. 2014). We have recently uncovered that individuals with DS show on average a 19-year increase in biological age when compared to chronologically age-matched controls of normal karyotype, however, the mechanism that causes this substantial difference is yet to be discovered. We hypothesize that these differences could be explained by the presence of a third copy of certain chromosome 21 genes, followed by the increased expression of proteins encoded by those genes. One candidate chromosome 21 gene is RUNX1 based on a recent GWAS study which found SNPs around this gene to be associated with human plasma IgG glycosylation (Klarić et al. 2020), and its extra copy has been discovered as an initiator of leukemogenic predisposition in DS (Nižetić and Groet 2012). We here used EBV-immortalized lymphoblastoid cell lines (LCLs) from an individual with DS and their disomic parent and sibling and treated the cells with a chemical inhibitor of the protein encoded by RUNX1. After treatment, the comparison of glycosylation profiles of IgG generated by these LCL cells revealed that inhibition of RUNX1 very significantly affected the glycosylation of IgG from LCLs derived from the disomic controls, yet no significant change in profile was observed in the cell-derived IgG of the person with DS. This finding complies with the aforementioned GWAS results and further implies that RUNX1 could be an important modulator of the general IgG profile in people with a normal number of chromosomes. Other mechanisms may prevail in skewing the glycan profiles in cells derived from people with DS.

Key words: Immunoglobulin G glycosylation, Down syndrome, RUNX1


Presentation number: MG 15

NEXT GENERATION SEQUENCING PANEL CUSTOMIZATION FOR LOSS AND REVERSIBILITY OF SENSE OF SMELL AND TASTE AFTER SARS-COV-2

Naida Lojo-Kadric, Jasmin Ramic, Nikolina Tomic, Belmina Saric, Lejla Pojskic

University of Sarajevo – Institute for genetic engineering and biotechnology, Laboratory for human genetics, Sarajevo, Bosnia and Herzegovina

Sensory-neural loss of taste and smell can occur as a result of the destruction of neuroepithelium by toxic inflammatory factors or due to genetic factors such as polymorphisms on olfactory or taste receptor genes. Partial or complete loss of smell (anosmia/hyposmia) and taste (hypogeusia/ageusia), with or without distorted perception of smell and taste (dysosmia/dysgeusia), has a broad differential diagnosis. Reversibility of loss of sense of smell and taste is possible in cases of inflammation, and can be sampled by various drugs, disorders and genetic factors. At the beginning of the COVID-19 pandemic, dysosmia and dysgeusia were not considered important symptoms for COVID-19. After the initial onset of the pandemic, several studies have reported taste and odor disorders in patients with SARS CoV-2 infection. Identification of dysosmia and dysgeusia may help in the early detection of SARS CoV-2 infection. There are approximately 400 OR genes (olfactory receptors) that have different pseudogenes, copy number variations, and single nucleotide (SNP) polymorphisms that can alter receptor responses, making the olfactory system and consequent dysosmia susceptible to various influences. In addition, there are 2 main families of genes for taste receptors that can influence dysgeusia, as well as several genes that are in SARS CoV 2 viral fusion cascade.  of this study is to design NGS panel which will elucidate main contributor genetic factors to dysosmia and dysgeusia. For the main panel design we used literature references and research on olfactory and taste receptor genes. For 144 genes that was our starting material we identified genome locations, sizes and SNP positions according to hg38 genome build. From that we filtered 69 genes that are main contributors, according to literature, for dysosmia/dysgeusia. After initial research, using DesignStudio by Illumina we checked the coverage which for our panel is 99% with 266 targets and 84,36 kb panel size with final count of 66 genes after we removed duplicates and low coverage target genes. Next step in study is validation and testing of panel to determine effectiveness of variant calling and efficiency of panel. The knowledge is applicable to further research on varying ethiology of loss of smell and taste and potential treatments.

Key words: NGS, dysosmia, dysgeuzia, COVID19


Presentation number: MG 16

NEUROOCULOCARDIOGENITOURINARY SYNDROME (NOCGUS) – CAUSED BY PATHOGENIC VARIANT IN THE WDR37 GENE – CASE REPORT

Višnja Tomac1,2, Silvija Pušeljić1,2, Martina Kos1,2, Ivana Serdarušić1, Nora Pušeljić1, Ema Poznić1, Paula Ivanšić1, Luka Perić3, Nika Pušeljić2

1Department of Pediatrics, Osijek University Hospital, Osijek, Croatia, 2Faculty of Medicine, University of Osijek, Osijek, Croatia, 3Department of Oncology, Osijek University Hospital, Osijek, Croatia

Neurooculocardiogenitourinary syndrome (NOCGUS) (OMIM# 618652) is a multisystem disorder described 2019 year, caused by heterozygous mutation in the WDR37 gene on chromosome 10p15, autosomal dominant inherritance. Characterized by neurological impairment with structural brain defects and seizures, poor feeding, poor postnatal growth, ocular anomalies, dysmorphic facial features, and variable skeletal, cardiac and genitourinary defects. Death in infancy may occur. We report a 7 years old boy, born in a second pregnancy (two other childrens is healthy) of nonconsanguineous parents, with intrauterine growth retardation, birth weight 2,5 kg. Dysmorphic features present from birth: hypertelorism, bilateral corneal opacity with keratoconus, gothic palate, depressed nasal bridge, ears lower laid poorly modeled, mild microretrognathia, thorax short “soft” chunky; hyperextensibility of all joints, knee contracture, hypoplastic scrotum, enlarged liver 3 cm, hypotension, impaired reflexes. Complementary treatment: developmental brain anomaly (cerebellar hypoplasia Dandy Walker variant, corpus callosum hypoplasia, cortical atrophy), with early onset of epilepsy; eye anomaly – congenital corneal opacity and keratoconus, congenital heart defect – ventricular septal defect, bicuspid aortic valve and aortic root dilatation. Inherited metabolic diseases are excluded, karyotyping and chromosomal microarray were normal. Pathogenic variant in the WDR37 gene was identified by whole exome sequencing (WES): 10-1126405-T-G; c.385T>G; heterozygous. Boy have profund intelectual dIsabillity (IQ 35) and global developement delay. Neurooculocardiogenitourinary syndrome (NOCGUS) is a very rare autosomal dominant genetic disorder The WDR37 is predicted to encode a 494-amino acid protein of unknown function. Data collected via publicly available databases suggest a broad pattern of expression for Wdr37 in mice with enrichment in ocular and brain tissues. There is no cure for NOCGUS, affected individuals need a team of specialized doctors for treating the various problems, which can occur.

Key words: Neurooculocardiogenitourinary syndrome, coloboma, epilepsy


Presentation number: MG 17

DNMT3B rs2424913 AS A RISK FACTOR FOR CONGENITAL HEART DEFECTS IN DOWN SYNDROME

Dijana Majstorović1, Anita Barišić2, Jadranka Vraneković2

1Juraj Dobrila University of Pula, Faculty of Medicine, Pula, Croatia, 2University of Rijeka, Faculty of Medicine, Department of Medical Biology and Genetics, Rijeka, Croatia

Congenital heart defects (CHDs) are the most common type of congenital malformations, present in approximately 40 to 50% of individuals with Down syndrome (DS). The most common CHDs in DS are septal defects. DNA hypomethylation is suggested to be associated with the development of CHDs in DS, particularly with septal defects. The DNA methylation pattern is established and maintained by DNA methyltransferases (DNMTs). The aim of this study was to assess the association between single nucleotide polymorphisms of DNMT genes and CHDs in DS individuals. The study was performed on 249 participants with DS, including 132 DS individuals with CHD (DSCHD+) and 117 DS individuals without CHD (DSCHD-). Genotyping of single nucleotide polymorphisms DNMT1 (rs2228611), DNMT3A (rs1550117), DNMT3B (rs1569686), and DNMT3B (rs2424913) was performed using PCR-RFLP method. Statistical significance was considered at P<0.05. The most common congenital cardiac defect among DSCHD+ participants was atrial septal defect (ASD), followed by a ventricular septal defect (VSD) and atrioventricular septal defect (AVSD). Statistically significant higher frequency of the DNMT3B rs2424913 CT and rs2424913 TT genotypes were observed in DSCHD+ group compared to DSCHD- group (p = 0.032; p = 0.011). Additionally, significance risk for CHD under the dominant genetic model (CC + CT vs TT) for DNMT3B rs2424913 (p= 0.011) was demonstrated. DNMT3B rs2424913 TT genotype, as well as the T allele, had a significantly higher frequency in DS individuals with ASD in comparison to DS individuals with other CHDs (p = 0.028; p = 0.018). Study results suggest that DNMT3B rs2424913 CT and rs2424913 TT genotypes, as well as the dominant genetic model of the same polymorphism, might be a possible predisposing factor for CHD in DS individuals, particularly in the ones with ASD.

Key words: congenital heart defects, DNA methyltransferase, Down syndrome, single nucleotide polymorphism


Presentation number: MG 18

SAUL-WILSON SYNDROME – COMPLEX SKELETAL ABNORMALITIES CAUSED BY PATHOGENIC VARIANT IN THE COG4 GENE – CASE REPORT

Silvija Pušeljić1,2, Višnja Tomac1,2, Nora Pušeljić1, Ema Poznić1, Martina Kos1,2, Ivana Serdarušić1, Paula Ivanšić1, Luka Perić3, Nika Pušeljić2

1Department of Pediatrics, Osijek University Hospital, Osijek, Croatia, 2Faculty of Medicine, University of Osijek, Osijek, Croatia, 3Department of Oncology, Osijek University Hospital, Osijek, Croatia

Saul-Wilson syndrome (OMIM # 618150) is a very rare disorder, at least 16 affected individuals have been reported in the scientific literature. Syndrom caused by mutation in the COG4 gene located at position 16q22.1, autosomal dominant inherritance. This gene provides instructions for making proteins known as the conserved oligomeric Golgi (COG) complex. Syndrom characterized by primordial dwarfism and other skeletal abnormalities, average adult height 107 centimeters. We report a 17 years old boy, body weight 19 kg; body height 96 cm; born in a second pregnancy (three other childrens is healthy) of nonconsanguineous parents, with intrauterine growth retardation, birth weight 2,1 kg. Dysmorphic features present from birth like arthrogryposis that disappear with growth, progeroid facial appearance, failure to thrive, lypodistrophy narrow nasal bridge with convex nasal ridge, prominent columella, mild micrognathia, blue sclerae. Skeletal findings include: profound short stature, clubfoot, short distal phalanges of fingers and toes. Flexion contractures of the knee joints, pseudoarthrosis, syringomyelia, thoracic scoliosis, lumbar hyperlordosis, coxa valga, skeletal dysplasia, pectus carinatum. Mild intelectual dIsabillity (IQ 65). Karyotyping and chromosomal microarray were normal. Pathogenic variant in the COG4 gene was identified by whole exome sequencing (WES): NM_015386.3: c.1546G> A (NP_056201.1: p.Gly516Arg), heterozygous. Saul-Wilson syndrome (SWS) is a very rare autosomal dominant genetic disorder The COG4 gene mutations that cause SWS result in production of an abnormal COG4 protein which is part of the COG complex, the transport of proteins between the Golgi apparatus and the endoplasmic reticulum is increased. It is unclear how this change in retrograde transport impairs bone growth and leads to the signs and symptoms of Saul-Wilson syndrome. There is no cure for SWS, affected individuals need a team of specialized doctors for treating the various problems, which can occur.

Key words: Saul-Wilson syndrome, dwarfism, skeletal abnormalities


Presentation number: MG 19

THE CONNECTION BETWEEN LEPTIN RECEPTOR GENE EXPRESSION, SERUM LEPTIN CONCENTRATION AND BODY MASS INDEX IN DIFFERENT MALIGNANT BREAST TUMORS DEPENDING ON LYMPH NODE METASTASES PRESENCE

Anita Matić1, Ivan Koprivčić2,3, Ksenija Marijanović3,4, Maja Tolušić Levak3,5, Robert Mujkić1, Vjekoslav Wertheimer2,3, Nikolina Lazić1

1Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 2Department of Orthopedics and Traumatology, Osijek University Hospital, Osijek, Croatia, 3Faculty of Medicine Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 4Clinical Department of Pathology and Forensic Medicine, Osijek University Hospital Osijek, Croatia, 5Department of Dermatology and Venereology, Osijek University Hospital, Osijek, Croatia

Previously we showed (Koprivić et al. Acta clinica Croatica) that obese females have a significantly higher level of leptin, regardless of the malignant breast tumor type. Observed through body mass index, the most significant differences in serum leptin levels are in the luminal B1 group. This study aimed to assess whether there was an association between leptin receptor gene (LEPR) expression and serum leptin concentration with body mass index (BMI), depending on the presence/absence of lymph node metastases. 53 females with malignant breast cancer were divided into two groups depending on the presence or absence of lymph node metastases (21 negatives and 32 positive lymph nodes). According to the St. Gallen Conference on breast cancer, carcinomas are divided into 5 subgroups according to molecular analysis and related to the expression profile of certain genes: Luminal A, Luminal B HER2 negative (LUM B1), Luminal B HER2 positive (LUM B2), HER2 positive and triple-negative subgroup. This division is based on estrogen, progesterone, and the expression of human epithelial growth factor. The positive or negative lymph node was specified with the tumor-node metastasis system classification AJCC (American Joint Committee on Cancer). LEPR gene level was determined by quantitative real-time PCR and serum leptin concentration by ELISA method. Each female was also measured BMI. Correlation analysis was calculated using Pearson’s correlation coefficient at 95% confidence of interval (SigmaPlot v11.2, USA). Statistically significant negative correlation was found between serum leptin level and gene LPRT expression (r=-0.451, P=0.0403) and significant positive correlation between BMI and serum leptin level (r=0.771, P=0.0000425) in group of females with absence lymph node metastases. In female malignant breast tumor groups with presence of lymph node metastases only significantly positive correlation was observed between BMI and serum leptin level (r=0.677, P=0.0000207). The association between the LPRT gene and serum leptin levels is more pronounced in malignant breast tumors with the absence of lymph node metastases. Regardless of the presence/absence of lymph node metastases, changes in serum leptin concentration depend on a woman’s BMI status.

Key words: breast tumor, leptin, leptin receptor, BMI, lymph node


Presentation number: MG 20

CONGENITAL BILATERAL HOANAL ATRESIA AS THE FIRST SIGN OF MUTATION IN THE CHD7 GENE

Ema Poznić1, Nora Pušeljić1, Višnja Tomac1,2, Silvija Pušeljić1,2, Martina Kos1,2, Ivana Serdarušić1, Paula Ivanšić1, Luka Perić3, Nika Pušeljić2

1Department of Pediatrics, Osijek University Hospital, Osijek, Croatia, 3Department of Oncology, Osijek University Hospital, Osijek, Croatia

CHARGE syndrome (OMIM # 214800) is a rare autosomal dominant genetic disorder that occurs in approximately 1 in 8,500 to 10,000 newborns, caused by mutation in the CHD7 gene located at position 8q12.2. Syndrom characterized by coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. We report a 6-month-old boy, born in a second pregnancy (one healthy child) of nonconsanguineous parents, with intrauterine growth retardation, premature birth at 34 weeks of gestation, birth weight 1,5 kg, as a life-threatening, premature, hypotrophic infant with multiple difficulties as part of an underlying congenital disease that may clinically respond to CHARGE syndrome. Dysmorphic features present from birth both nostrils impassable by probe, dolichocephalic head, high forehead, depressed nasal bridge, blepharophimosis, thin upper lip, gothic palate, microretrognathia, generalized hypotonia, laryngotracheomalacia, callosum corpus hypoplasia, ventricular septal heart defect, cryptorchidism. Twice time surgically treated bilateral congenital hoanal atresia Karyotyping is normal. Pathogenic variant in the CHD7 gene was identified by whole exome sequencing (WES): 8-61757422-GT-G; c.4852del; heterozygous. Boy have global developement delay. CHARGE syndrome is a rare autosomal dominant genetic disorder and in hoanal atresia it is necessary to think of CHARGE as the cause The CHD7 gene provides instructions for making a protein that regulates gene expression by a chromatin remodeling. Most mutations in the CHD7 gene lead to the production of an abnormal CHD7 protein that is broken down prematurely. Shortage of this protein is thought to disrupt chromatin remodeling and the regulation of gene expression.

Key words: CHARGE syndrome, choanal atresia, heart defect


Presentation number: MG 21

EXPRESSION PATTERN OF APOPTOTIC INDUCING FACTOR IN THE INNER EAR DEVELOPMENT OF YOTARI (DAB1 -/-) AND WILD TYPE MICE

Azra Kazazić1, Nela Kelam2,3, Anita Racetin2,3, Natalija Filipović2,3, Yu Katsuyama4, Katarina Vukojević1,2,3

1Department of Anatomy, University of Mostar, School of Medicine, Mostar, Bosnia and Herzegovina, 2Department of Anatomy, Histology and Embryology, University of Split School of Medicine, Split, Croatia, 3Center for Translational Research in Biomedicine, University of Split School of Medicine, Split, Croatia, 4Department of Anatomy, Shiga University of Medical Science, Ötsu, Japan

DAB1-protein deficiency was investigated on the inner ear development of yotari in comparison to wild-type (wt) mice by expression of apoptotic inducing factor (AIF) at mice embryonic E13.5 and E15.5 in order to examine caspase independent apoptotic pathway. The spatial and temporal immunofluorescence expression pattern of AIF was determined by calculating area percentage covered by positive signal in the epithelium and mesenchyme of cochlear and semicircular ducts. Data were analyzed by t-test and were presented as a mean±SD.  AIF expression in the epithelium of cochlear and semicircular duct were significantly higher in wt in comparison to yotari mice. The highest AIF expression was at E15.5 in the epithelium of cochlear duct, but this expression was twofold lower than in wt mice. AIF expression in mesenchyme of the cochlear and semicircular ducts were always statistically higher in wt in comparison to yotari, except for higher AIF expression in the cochlear duct of wt at E15.5 in comparison to yotari mice. Our results emphasize the relevance of AIF during development of vestibular and cochlear functions where they can serve as potential therapeutic targets in impairments of the inner ear.

Key words: apoptotic inducing factor (AIF), inner ear, yotari and wild-type mice, expression


Presentation number: MG 22

INVOLVEMENT OF EPITHELIAL TO MESENCHYMAL TRANSITION FACTORS DURING THE HUMAN EYE EMBRYOGENESIS AND TUMORIGENESIS

Josipa Marin Lovrić1,2, Natalija Filipović3, Ljubo Znaor1,2, Anita Rančić1,2, Josko Petričević4, Nenad Kunac5, Violeta Soljić6,7, Mirna Saraga-Babić3, Katarina Vukojević3,6,8,9

1Department of Ophthalmology, University of Split School of Medicine, Split, Croatia, 2Department of Ophthalmology, University Hospital of Split, Split, Croatia, 3Department of Anatomy, Histology and Embryology, University of Split School of Medicine, Split, Croatia, 4Department of Pathology, University of Mostar, School of Medicine, Mostar, Bosnia and Herzegovina, 5Department of Pathology, University Hospital of Split, Split, Croatia, 6Department of Histology and Embryology, University of Mostar, School of Medicine, Mostar, Bosnia and Herzegovina, 7University of Mostar, Faculty of Health Studies, Mostar, Bosnia and Herzegovina, 8Department of Anatomy, University of Mostar, School of Medicine, Mostar, Bosnia and Herzegovina, 9Center for Translational Research in Biomedicine, University of Split School of Medicine, Split, Croatia

The expression pattern of cytokeratin-8, vimentin, nestin, beta-tubulin, HSP70 and syndecan-1 markers was analyzed in histological sections of 8th week developing and postnatal human eye, in retinoblastoma and different uveal melanomas to establish their involvement in epithelial to mesenchymal transition. Tissue sections were examined by double immunofluorescence to abovementioned markers and the area percentage of positive signals was evaluated quantitatively and semi-quantitatively. The one-way ANOVA followed by Tukey’s posthoc test was used for statistical analysis. Vimentin immunoreactivity characterized retinal and/or choroidal cells in healthy and tumorous tissues: expression was lower in developing retina and retinoblastoma, while it was high in epitheloid and spindle melanoma. Beta-tubulin and HSP-70 expression was highest in tumor tissue of retinoblastoma, epitheloid and mixoid uveal melanomas. Cytokeratin-8 was observed only in development and rarely in the choroid of mixoid melanoma. Nestin immunoreactivity was highest in the retinoblastoma and spindle melanoma, and missing in epitheloid melanoma, while sindecan-1 had highest expression in epitheloid and mixoid melanoma. Their differential expression appeared between types of melanomas. The differences in the expression pattern of factors involved in epithelial to mesenchymal transition correlate with the origin and stage of cell differentiation of tissue samples. The balanced expression pattern is required for both human eye development and eye tumorigenesis. Therefore, understanding of their involvement and interplay is important for possible new therapeutical targets based on epithelial to mesenchymal transition underlined in developmental eye plasticity and neoplasm.

Key words: human eye, embryogenesis, tumorigenesis, epithelial to mesenchymal transition

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Published: June 21st, 2022;

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