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Genetska testiranja kod pacijenata s neurorazvojnim poremećajima vrlo su važna za postavljanje konačne dijagnoze. Prema trenutnim smjernicama prve metode izbora uključuju komparativnu genomsku hibridizaciju na mikropostroju te genetsko testiranje fragilnog X sindroma. Cjeloeksomsko sekvenciranje koje omogućuje detekciju jednonukleotidnih varijanti u kodirajućim regijama gena predstavlja sljedeći dijagnostički korak. Dijagnostički prinos komparativne genomske hibridizacije na mikropostroju raste do 15%, a cjeloeksomskog sekvenciranja čak do 40%. Međutim, kod velikog broja pacijenata (čak do 50%) etiologija neurorazvojnih poremećaja ostaje nerazjašnjena. Danas su poznati mnogi genetski mehanizmi koje nije moguće ustanoviti rutinskim dijagnostičkim metodama, a uključuju varijante broja kopija u nekodirajućim regulacijskim DNA regijama, varijante broja kopija koje utječu na strukturu i funkciju topoloških domena, duboke intronske varijante u kodirajućim genima kao i metilacijske obrasce u genomu. Navedene genske promjene moguće je detektirati novijim tehnologijama kao što je cjelogenomsko sekvenciranje i mapiranje topoloških domena (Hi-C sekvenciranje). Cjelogenomskim sekvenciranjem moguće je odrediti i precizne točke loma u naoko balansiranim kromosomskim razmještanjima. Uvođenje cjelogenomskog sekvenciranja u rutinsku dijagnostiku genetskih bolesti svakako bi povećalo dijagnostički prinos i omogućilo postavljanje dijagnoze za velik broj pacijenata s neurorazvojnim poremećajima. S napredovanjem tehnologije i sve većom dostupnošću cjelogenomskog sekvenciranja, u skoroj budućnosti se očekuje i povećanje genomskih baza koje bi omogućile interpretaciju velike količine podataka koju ova metoda generira, čime bi se ubrzalo uvođenje ove moćne metode u rutinsku medicinsku skrb kod pacijenata s genetskim poremećajima., Genetic testing in patients with neurodevelopmental disorders is very important for making a final diagnosis. According to the current guidelines, the first line methods include Microarray-based Comparative Genomic Hybridization and genetic testing of Fragile X Syndrome. Whole-exome sequencing, which allows detection of single-nucleotide variants in the coding regions of genes, represents the next diagnostic step. The diagnostic yield of Comparative Genomic Hybridization is up to 15%, while for whole-exome sequencing it increases up to 40%. However, in a large number of patients (up to 50%), the etiology of neurodevelopmental disorders remains unexplained. Even though there are many familiar genetic mechanisms, some of them cannot be established by routine diagnostic methods, such as copy number variants in non-coding regulatory DNA regions, copy number variants that affect structure and function of topologically associating domains (TADs), deep intronic variants in coding genes as well as genome-wide methylation patterns. The aforementioned genetic changes can be detected with newer technologies such as whole-genome sequencing and mapping of TADs (Hi-C sequencing). With whole-genome sequencing it is possible to determine precise breakpoints in apparently balanced chromosomal arrangements. Therefore introduction of whole-genome sequencing in the routine diagnostics of genetic diseases would certainly increase the diagnostic yield and enable setting up diagnosis for a large number of patients with neurodevelopmental disorders. With the advancement of technology and the increasing availability of whole-genome sequencing, a rise in the amount of available data in genomic databases is expected in the near future. This would allow the interpretation of the large amount of data generated by this method and accelerate the introduction of this powerful method into routine medical care for patients with genetic disorders.

Cilj ove doktorske disertacije bio je istražiti molekularnu epidemiologiju HIV-1 infekcije među prethodno neliječenim HIV-om zaraženim osobama iz Hrvatske. U istraživanje je uključeno 403 ispitanika, odnosno 92,4% svih HIV-om zaraženih osoba koje su ušle u kliničku skrb u Hrvatskoj tijekom vremenskog perioda od 2014. do 2017. godine. Prevalencija primarne rezistencije HIV-1 na inhibitore reverzne transkriptaze (RTI), proteaze (PI) i integraze (InSTI) analizirana je tehnologijom sekvenciranja po Sangeru. Ukupna prevalencija primarne rezistencije na antiretrovirusnu terapiju (ART) iznosila je 16,4%. Prevalencija rezistencija prema pojedinim klasama lijekova, nukleozidnim RTI (NRTI), nenukleozidnim RTI (NNRTI), PI i InSTI iznosila je 11,4%, 6,7%, 2,5% i 1%. U istraživanju je utvrđena i primarna rezistencija na tri klase lijekova kod 2,2% ispitanika. Uz to, 48 ispitanika je nasumično odabrano za analizu mutacija povezanih s primarnom rezistencijom na ART pomoću tehnologije sekvenciranja nove generacije, a dodatne mutacije su pronađene u 6 uzoraka. Filogenetska analiza pokazala je visok postotak klasteriranja (347/403, 86,1%) i identificirala aktivno širenje više mutacija povezanih s rezistencijom u Hrvatskoj, čak i širenje rezistencije na tri klase lijekova. Najveći klaster s mutacijom povezanom s primarnom rezistencijom na ART bio je T215S klaster, formiran od 53 ispitanika, a filodinamičkom analizom je njegov razvoj procijenjen u 1992. godini. Rezultati ove studije ukazuju na kontinuiranu potrebu za nadziranjem primarne rezistencije HIV-1 i razvoja transmisijskih klastera u Hrvatskoj., The aim of this study was to investigate the molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia. Study included 403 persons, representing 92,4% of all HIV-1 positive individuals entering clinical care in Croatia in 2014-2017. The prevalence of transmitted drug resistance (TDR) to reverse transcriptase inhibitors (RTI), protease inhibitors (PI) and integrase strand-transfer inhibitors (InSTI) was assesed by Sanger sequencing. Overall prevalence of TDR was 16,4%. According to drug classes, resistance to nucleoside RTI (NRTI), non-nucleoside RTI (NNRTI), PI and InSTI was found in 11,4%, 6,7%, 2,5% and 1% of individuals, respectively. Triple-class resistance was determined in 2,2% of individuals. In addition, 48 individuals were randomly selected for next generation sequencing and additional TDR mutations were detected in 6 samples. Phylogenetic analysis showed high rate of clustering (347/403, 86,1%) and identified forward transmission of multiple TDR mutations in Croatia, even that of triple-class resistance. The largest cluster harbouring TDR consisted of 53 persons with T215S mutation and phylodinamic analysis estimated to originate in the year 1992. This data shows a continuing need for pre-treatment HIV-1 resistance testing and surveillance of local transmission networks in Croatia.

Purpose: Phylogenetic analysis is a useful tool for identification of HIV-1 transmission clusters and analysis of biological characteristics of individual clusters. The aim of the study was to determine and characterize transmission networks that are responsible for the forward spread of HIV-1 infection and resistant variants in Croatia. Method: We analyzed 403(95.9%) of 428 newly HIV-diagnosed persons who entered clinical care at the University Hospital for Infectious Diseases, Zagreb. The entire protease HIV-1 gene (PR, codons 1–99) and a part of the reverse transcriptase HIV-1 gene (RT, codons 1–240) were sequenced by using a validated in-house method. Mutations were determined by using Surveillance Drug Resistance Mutation list. HIV subtype was determined with Rega HIV-1 subtyping tool, version 3.0. Sequences subtyped as B were selected for phylogenetic inference. For each sequence in the dataset 10 closest sequences were determined with BLAST search. Phylogenetic trees were constructed with PhyML 3.0, while the Figtree version 1.4.3 was used for tree visualization. Transmission cluster were defined as sequences ≥3 patients from Croatian cohort with the approximate likelihood ratio test value >0.90. Results: Subtype B was found in 368 (91%) of patients. The overall prevalence of transmitted drug resistance (TDR) was 16.4% (n=66/403). The most prevalent TDR patterns were T215S, 7.2% (n=29/403) ; K101E, 3.5% (n=14/403) ; T215S + L210W, 2.2% (n=9/403) ; V32I+I47V+T215E/D+L100I+K103N, 1.9% (n=8/403) ; M41L+T215L, 0.7% (n=3/403). Phylogenetic analysis identified 19 local transmission clusters, of which 5 (26%) clusters were responsible for the forward spread of resistant HIV-1 viral strains. Patients in clusters (n=347) were more frequently MSM (319/358 vs. heterosexuals 24/39, p

Croatia has a centralized system of HIV clinical care and all patients are treated at University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”, Zagreb (UHID). Previous national study showed a high prevalence of transmitted drug resistance (TDR) of 21.6% (in the period 2006 to 2008) (1). The study analyzed primary resistance to reverse transcriptase (RTI) and protease inhibitors (PI). Our recent study confirmed that TDR is still very high in Croatia (23.4%, analysed for the time period 2014-2015) (2). Analysis of primary resistance to integrase strand transfer inhibitors (INSTI) has not yet been examined in Croatia, however during 2016-2017 there were 3 clinical cases of INSTI resistance among INSTI-treated HIV-1 patients. With the appearance of first clinical cases that confer resistance to INSTI, there is a possibility for circulation of INSTI-resistant HIV-1 among newly diagnosed HIV-1 patients. Therefore, in this study the prevalence of TDR to INSTI was examined in a cohort of newly diagnosed HIV-1 patients from Croatia.

The geographical distribution and prevalence of HIV-1 subtypes in Europe are highly heterogeneous. Subtype B is predominant in Western and Central Europe but non-B subtypes, introduced mainly via migration, are also present in the region. Croatia is a small Central European country with distribution of HIV-1 subtypes similar to that of other European countries dominated by subtype B. Several studies on HIV-1 subtype distribution have been published in Croatia so far that show that the most common HIV-1 subtype is subtype B. Molecular analysis of HIV subtypes in period 2001-2003 showed a high prevalence of subtype B (>74%) with non-B subtypes found only in heterosexuals. A study on transmitted-drug resistance (TDR) in newly diagnosed HIV-infected patients conducted in the period 2006-2008 showed a high prevalence of subtype B among MSM population with only 11 % of patients infected with non-B subtypes. Subtype B was also confirmed as predominant in respond-driven sampling (RDS) study among MSMconducted in 2006 and 2010. The aim of this study is to show a more recent data on of HIV-1 subtype distribution in newly diagnosed patients in Croatia and compare it with previous years.

Phylogenetic analysis is a useful tool for identification of HIV-1 transmission clusters (TC) and analysis of biological characteristics of individual clusters. The aim of the study was to determine and characterize transmission networks that are responsible for the forward spread of HIV-1 infection and resistant variants in Croatia.

Croatia has a centralized system of HIV clinical care and all patients are treated at University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”, Zagreb (UHID). Previous national study by Grgic et al. showed a high prevalence of transmitted drug resistance (TDR) of 21.6% (in the period 2006 to 2008), mainly attributed to the transmission clusters among MSM characterized by the presence of T215S mutation (1). In order to continue national TDR surveillance, we analyzed the prevalence of TDR mutations for the time period 2014-2015. Included were newly diagnosed, treatment-naive patients who entered clinical care at UHID during the study period.

Croatia has a centralized system of HIV-1 care and all patients are treated at University Hospital for Infectious Diseases “Dr.Fran Mihaljević”. HIV drug- resistance testing is routinely performed for all patients experiencing virological failure and for selected therapy-naive patients. Results of our previous national study showed substantially higher prevalence of transmitted drug resistance mutations(TDRM)of 21.6%(in the period 2006 to 2008, compared with European data, with the prevalence of 8.4%(in the period 2002-2005.Moreover, the study by Grgic et al. determined the highest risk factor for HIV transmission in the group of men who have sex with men(MSM(80/118 patients, 67.8%), who accounted for 76.9% of all transmitted drug resistance cases.Since the acute/recent phase of infection represents the most opportune time to detect transmitted drug resistance(TDR, the goal of the study was to determine the prevalence of TDRM in therapy-naive patients with recently acquired HIV infection, who entered clinical care at our center in the period 2014-2016. Additionally, the evolutionary relationship between nucleotide sequences of patients who carried T215S primary resistance mutation and their possible interconnection with the local T215S transmission cluster was assessed.