Presentation number: MG 28
KNOWLEDGE AND ATTITUDES ON GENETIC TESTING IN CROATIAN POPULATION: A PRELIMINARY STUDY
Ana Banovac, Livia Sliskovic, Ivan Jerkovic
University Department of Forensic Sciences, University of Split, Split, Croatia
Aim was to investigate the knowledge and attitudes about genetic testing in the Croatian general population and determine the factors affecting their willingness to participate in different types of genetic testing. We conducted a cross-sectional online survey from 6 to 14 March 2022 on a sample of 215 adults (72.1% females, median age = 31). The survey questionnaire included general demographic data, data on blood donation, and diagnosis of severe diseases in relatives. We included questions regarding knowledge on genetic testing (e.g., the meaning and availability) and created two scales measuring participants’ attitudes toward genetic testing. The first scale examined their attitudes and willingness to participate in genetic testing, while the second one explored fear related to involvement in such activities. Participants demonstrated on average relatively positive attitudes and higher levels of willingness to participate in genetic testing (total score = 32.05/40; 95%CI 31.03-32.96) and moderate levels of fear related to involvement in such activities (total score = 10.94/20; 95%CI 10.35-11.53). Participants’ sex (P = 0.048), religion (P = 0.047), and reported knowledge (P < 0.001) significantly contributed to their attitudes and willingness to participate in such activities, while their fear of involvement was related to political affiliation (P = 0.012). Croatian population shows openness toward genetic research and testing that can be affected by their sex, religion, knowledge, and political affiliation. To obtain better insight into those factors, future studies should be conducted on a larger and more comprehensive sample.
Key words: genetic testing, attitude, knowledge, Croatian population, personalized medicine
Presentation number: MG 29
FREQUENCY OF HLA DQ2.5 AND HLA DQ8 GENOTYPE IN CLINICAL PATIENTS WITH IBD SYMPTOMS OVER FOUR-YEAR PERIOD IN POPULATION OF BOSNIA AND HERZEGOVINA
Selma Durgut1, Lana Salihefendić1, Ivana Čeko1, Naida Mulahuseinović1, Adna Ašić2, Adis Kandić1, Rijad Konjhodžić1
1Alea Genetic Centre, Health Institute Alea Dr. Kandić, Sarajevo, Bosnia and Herzegovina, 2Department of Genetics and Bioengineering, International Burch University, Sarajevo, Bosnia and Herzegovina
Celiac disease is an autoimmune disorder affecting the inflammation of the small intestine, triggered by the gluten consumption. Predisposition is determined by the HLA II DQA and DQB genes. Symptoms can be very unpleasant for individuals, but since they may disappear with gluten-free diet, an early celiac diagnosis is very important. Aim of this study is to determine the distribution of HLA genotype frequency in representative samples of Bosnian-Herzegovinian population with IBD symptoms. For this study a total of 170 patients with clinical IBD symptoms which were tested for HLA-DQ2.5 or HLA-DQ8 genotype, were observed. Clinical data were collected from medical histories of patients. All patients underwent HLA genotyping in ALEA Genetic Center from 2018 to the end of March, 2022. Buccal swabs or blood samples were used as the DNA source. For the testing, Celiaclear (molGENTIX SL, Spain) reagents were used according to manufacturer’s instructions. To detect HLA genotype, fragmental analysis was performed on the SeqStudio Genetic Analyzer. This study showed that there was a strong association between results of HLA-DQ2.5/DQ8 molecular testing and clinical manifestations of patients. This molecular analysis was recommended to patients based on their clinical features. Patients mainly reported: abdominal pain, diarrhea, bloating, gas, constipation, fatigue, reduced appetite and weight loss. Correlation of these symptoms with celiac disease was confirmed by HLA molecular typing, where results themselves coincided with clinical manifestations for most of the patients. During the study, it was noticed that the HLA-DQ2.5 was more frequent genotype in the population of Bosnia and Herzegovina over four year period. Clinical manifestation can significantly indicate the type of molecular analysis. HLA molecular typing for celiac disease is an important parameter for the discrimination of individuals genetically susceptible to celiac disease.
Key words: celiac disease, HLA genotype, molecular testing, IBD symptoms
Presentation number: MG 30
CHALLENGES IN OBTAINING HIGH-QUALITY DATA FROM A CUSTOM-MADE PANEL FOR THE NEXT GENERATION SEQUENCING (NGS) USING ION TORRENT GENESTUDIOTM S5 PLATFORM
Lana Salihefendić1,2, Adna Ašić1, Ivana Čeko2, Dino Pećar2, Larisa Bešić1, Naida Mulahuseinović2, Amra Džuho1, Laura-Severina Köhn1, Rijad Konjhodžić2
1International Burch University, Department of genetics and bioengineering, Sarajevo, Bosnia and Herzegovina, 2Alea Genetic Center, Sarajevo, Bosnia and Herzegovina
Aim was the sequencing procedure optimization of a total of 16 human genes and their regulatory regions from 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina. Selected genes were found to be potentially associated with differential immunological COVID-19 response according to previously published data. Methods: DNA isolation from whole blood was performed using QIAamp® DNA Mini Kit, as per the manufacturer’s instructions. Next Generation Sequencing was conducted on Ion Torrent GeneStudioTM S5 platform. Library preparation was done using Ion AmpliSeqTM Library Kit Plus and was optimized for low-quality DNA. In addition, three samples were subjected to clinical exome sequencing using the TruSight One Sequencing Panel (Illumina, San Diego, CA). NGS was optimized through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool that showed poorer amplification results. Primer pool 1 obtained results for all of the 60 patients, while pool 2 obtained results for a total of 48 patients, including three clinical exome sequences. The analysis was not limited by the quality of collected samples and DNA, but by the quality of custom-made primers from pool 2. Separating the two primer pools allowed for complete results when it comes to primer pool 1 and partial completion of results with primer pool 2.
Key words: COVID-19, next-generation sequencing, Ion Torrent GeneStudioTM S5, immunological response
Presentation number: MG 31
EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) MOLECULAR TESTING IN HISTOLOGY AND CYTOLOGY SPECIMENS
Clinical Hospital Dubrava, Clinical Department of Pathology and Citology, Laboratory for Immunohistochemistry and Molecular pathology, Zagreb, Croatia
Lung cancer is a leading cause of cancer related deaths in the world. Adenocarcinoma is one of the most commonly diagnosed subtypes of non-small cell lung cancer. Nowadays, as medicine advances, treatment is based on the molecular characteristics of certain types of cancer. The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases. EGFR sequencing at the Clinical Hospital Dubrava is performed using the Idylla™ EGFR mutation test on the Biocartis Idylla system. It is an in vitro polymerase chain reaction (PCR) test that is the most commonly used method for detecting EGFR mutations and is key evidence for investigating possible gene therapy in patients with non-small cell lung cancer. Idylla™ EGFR mutation test is a diagnostic test for qualitative detection of exon 18 (G719A/C/S), exon 21 (L858R, L861Q), exon 20 mutations (T790M, S7681), exon 19 deletion and exon 20 insertions. From September 2020 until March 2022, a total of 89 samples for EGFR mutation were tested. The diagnosis of lung adenocarcinoma was made by histological and cytological examination. There were 59 histological sections (66.3%) and 30 cytological smears (33.7%). The mutation was found in 23 (25.8%) samples, 11 (47.8%) in histological and 12 (52.2%) in cytological smears. The most common mutation was deletion in exon 19. The second most common mutation is L858R on 4 histological samples. The L861Q mutation was confirmed in 2 metastatic adenocarcinomas on cytological smears. The insertion in exon 20 was confirmed on 1 cytological smear, as well as the T790M mutation and the deletion in exon 19. The G719A/C/S mutation was confirmed in 2 cytological samples, while the L858R and L861Q mutations were confirmed in 2 cytological smears. Biocartis Idylla™ is simple system for rapid detection of relevant mutations. Due to the high sensitivity, the system gave positive results already at 20% share of tumor cells. With fast handling and results ready in 150 minutes, this method accelerated the analysis and the possibility of applying targeted therapy. Lung adenocarcinoma patiens with detected EGFR mutations are treated with tyrosine kinase inhibitors erlotinib, gefitinib and crizotinib. A bigger issue is becoming resistance to these drugs, so the priority is the development of new target drugs.
Key words: adenocarcinoma, EGFR, histology, citology
Presentation number: MG 32
SARS-COV-2 VIRUS: COMPARISON OF MANUAL AND AUTOMATIZED RNA EXTRACTION TECHNIQUES FROM NASOPHARYNGEAL SAMPLES
Ivana Čeko, Merima Smajlhodžić-Deljo, Alma Suljić, Lana Salihefendić, Dino Pećar, Adis Kandić, Rijad Konjhodžić
ALEA Genetic Center, Health Institute ALEA Dr. Kandić, Sarajevo, Bosnia and Herzegovina
SARS-CoV-2 virus caused the COVID-19 pandemic and the fast spread of SARS-CoV-2 (COVID-19) has created the need for fast diagnostic testing. Reliable protocols for viral RNA extraction and amplification are crucial for the detection of SARS-CoV-2 virus. The aim of this study was to compare seven RNA extraction techniques, of which two were automatized RNA extraction techniques and five manual RNA extraction techniques. The detection of extracted RNA was validated by LabGunTM COVID-19 PCR Kit (Ref CV9017B) from LabGenomics Co., Ltd targeting RdRp gene, based on their Ct value. Thirty clinical nasopharyngeal samples were collected in ALEA Genetic Center for the comparison of different techniques for RNA extraction. Twenty four nasopharyngeal samples were positive samples and six negative samples were used as a negative extraction control. The Bio-Rad CFX96 Touch Real-Time PCR Detection System with the LabGunTM COVID-19 Assay was used as a molecular detection technique for SARS-CoV-2. T-test was used for the comparison of different extraction techniques. Values with p<0.05 were considered statistically significant. Results show that most of these techniques meet the basic requirements for RNA extraction. Two extraction techniques have been chosen with optimal results in all of the parameters (cost effectiveness, RNA yield and RT-PCR results).
Key words: SARS-CoV-2 virus, RNA extraction, Real-time PCR
Presentation number: MG 33
COMPARATIVE ANALYSIS OF SARS-COV-2 DETECTION KITS
Naida Mulahuseinović, Lana Salihefendić, Selma Durgut, Enis Kandić, Rijad Konjhodžić
ALEA Genetic Center, Department for Molecular Biology, Sarajevo, Bosnia and Herzegovina
SARS-CoV-2 is a coronavirus that causes a respiratory disease, COVID-19. For COVID-19 testing, real-time PCR is considered gold standard and therefore many commercial SARS-Cov-2 detection kits are available. Rapid and accurate diagnostic tests are essential for controlling the COVID-19 pandemic. Aim of the study is to determine diagnostic values of 10 different commercially available SARS-CoV-2 detection kits, based on their Ct value. For this study thirty clinical nasopharyngeal samples were collected in ALEA Genetic Center. Twenty four of them were positive, while six were negative and used as a negative control. Positive samples were selected based on the day when first symptoms appeared. RNA was extracted using the same extraction method for all samples. For amplification and comparison of detection kits, the same RT- PCR instrument was used. Accuracy, sensitivity, specificity and Cohen’s kappa coefficient were estimated to evaluate diagnostic values of the tested kits. This study showed that all kits showed 100% specificity. Accuracy, sensitivity and kappa coefficient varied among examined assays. Based on clinical features, LabGunTM COVID-19 Assay by LabGenomics proved to be the most sensitive, the most accurate and most specific. Therefore, this assay was used as a reference kit. If things from practice are taken into account, accuracy and reliability of the tested commercial kits can vary compared to those obtained in this study where results were based on ideal functioning of the kits. When choosing the convenient commercial SARS-CoV-2 detection kit using RT-PCR method, many parameters need to be considered.
Key words: SARS-CoV-2, SARS-CoV-2 detection kits, real-time PCR
Presentation number: MG 34
PRENATAL RHD GENOTYPING BY NIPT METHOD: CROATIAN EXPERIENCE
Jasna Bingulac-Popović, Irena Jukić
Croatian Institute of Transfusion Medicine, Department of Molecular Diagnostics and Medical Department, Zagreb, Croatia
Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive prenatal fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The preanalytical factors, diagnostic performance, and results of validated in house RT-PCR method are investigated. Materials and methods RHD genotyping of 205 RhD negative pregnant women in 12-36th week of gestation was performed in period between 2015 and 2019. QIA symphony SP DSP Virus Midi Kit was used with modification to obtain optimized cffDNA yield on QIA symphony SP device (Qiagen, Germany). Fragments of RHD exons 7, 10 and later exon 5 were RT-PCR amplified. As internal controls, fragments SRY or RASSF1A gene with β-actin genes digested with BsTUI were used. Results 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes were detected. One inconclusive result (0.5%) was due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. Our method enables detection of fetal D variant inherited from the father in RHD*04.04/*01N.01 genotype. When compared to newborn’s RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity, 95.08% specificity and 98.48% diagnostic accuracy. The negative and positive predictive test values were 100% and 97.86%, respectively. Conclusion Careful sample handling, automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and internal controls of SRY, RASSF1A fragments represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly decontamination protocol is required. Introduction the mandatory NIPT RHD screening for whole country requires a fully automated process on platform used only for this method. This would shorten the time to results, allow better standardization, and reduce cross-contamination risk.
Key words: non-invasive prenatal RHD genotyping, cell-free fetal DNA, anti-D immunoprophylaxis.
Presentation number: MG 35
PLACENTAL PATHOLOGY CHANGES OF THE THIRD TRIMESTER PREGNANT WOMEN FROM COVID-19: HISTOLOGICAL, BIOMOLECULAR AND IMMUNOHISTOCHEMICAL STUDY
Emiliano Maresi1, Gaia Morello1, Elisabetta Orlando1, Giovanni De Lisi1, Clio Bilotta1, Giuseppe Davide Albano1, Antonina Argo1, Ada Maria Florena1
1PROMISE Department, University of Palermo, Palermo, Italy
The present study aimed to report the analysis of histopathological, immunohistochemical, and molecular of a large series of placentas from SARS-CoV-2-positive mothers observed at a “San Giovanni di Dio” Hospital in Agrigento during the pandemic and to compare them with a control group to highlight any histopathological alterations attributable to SARS-CoV-2. Regarding placental disease in SARS COV-2 virus-positive pregnant women, only case reports or small, limited case series were reported during the pandemic. Twenty-one placentas from the third-trimester pregnancy women were studied. Twenty-one selected received singleton third-trimester placentas consecutively from SARS-CoV-2-negative women from the same time period were reviewed for comparison. All patients were cured, and no clinical or serological evidence pointed to vertical transmission of SARS-CoV-2. In SARS CoV-2 virus-positive pregnant women were only observed aspecific lesions: Maternal VascularMalperfusion (MVM) were present in 19 (90.4%) cases; Fetal Vascular Malperfusion (FVM) lesions occurred in 20 (95.2%) cases; Maternal/Fetal Inflammatory Response (MFIR) was observed in 3 (14.2) cases. In 17 cases (80%), MVP and FVM were associated; in 2 cases(10%), FVM and MFIR were associated; in 1 (5%) case, the MVM, FVM, and MFIR occurred together; in 1 (5%) case MVM occurred alone. A comparison of placental lesions between SARS COV-2 virus-positive pregnant women and the control group showed a statistically significant difference (p-value: 0.0089). In no cases does biomolecular and immunohistochemical analysis (RNASCOPE with probe SARS CoV-2; anti-spike protein) demonstrate viral mRNA or spike protein. The preterm newborns were significantly present (p-value: 0.0048) in pregnant women virus-positive during the third trimester of pregnancy “remote” from delivery. We found no evidence of vertical transmission and adverse maternal-fetal outcomes in the placentas of third trimester COVID-19 pregnancy women, which provided further information for the clinical management of those women in the third trimester. However, further studies are still needed for patients with infections in different stages of gestation, especially in the first and second trimesters.
Key words: Placental pathology, SARS-CoV-2 infection, vertical transmission, immunohistochemistry