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Prirojene napake veljajo za enega izmed glavnih vzrokov neonatalne umrljivosti v današnjem razvitem svetu. Med najpogostejše spadajo orofacialne shize in prirojene srčne napake. Orofacialna shiza je prirojen razcep ustnice, neba ali čeljustnega grebena, lahko pa gre za kombinacijo vsega. Predstavlja velik estetski in funkcionalni problem, saj v različnih oblikah in stopnjah združuje nosno in ustno votlino. Prirojena srčna napaka pa nastane kot posledica motenega razvoja srca in/ali velikih žil. Pojavlja se v različnih segmentih srca in jih povezuje med sabo. Kljub veliki razširjenosti in pomembnosti orofacialnih shiz in prirojenih srčnih napak, je genetsko ozadje vzroka njunega sočasnega pojava slabo poznano. Število genov, vpletenih v njun razvoj, je namreč zelo veliko. Cilj magistrske naloge je tako bil izboljšati genetsko diagnostiko bolnikov z orofacialnimi shizami in prirojenimi srčnimi napakami, saj lahko le z razumevanjem in prepoznavo mehanizmov njunega sočasnega pojava, delujemo preventivno in takšne primere hitro obravnavamo. V magistrski nalogi smo zato izvedli sistematični pregled literature, kjer smo iskali vse poročane genetske spremembe pri bolnikih s sočasno prisotnostjo orofacialne shize in prirojene srčne napake. Po postavitvi iskalnega profila smo v bazi podatkov PubMed poiskali ustrezno literaturo. Definirali smo vključitvene kriterije in nato obravnavali tiste študije, ki so postavljenim kriterijem ustrezale. Takšnih študij je bilo 210, v sklopu katerih smo identificirali 611 primerov posameznikov z več kot 150 različnimi genetskimi spremembami in s sočasno prisotnostjo obeh fenotipov. Najpogostejša genetska sprememba izmed vseh je bila mikrodelecija 22q11, ki smo jo zaznali kar 364-krat. Ostale pogoste genetske spremembe so bile mutacije v genih PGM1, MEIS2, STAG2, BMP2, PGAP3 in RPL5, delecija 15q14, terminalne delecije 4q in trisomija 22, 10 in 9. Nekatere genetske spremembe so pripadale tudi znanim sindromom, ki so jih že uspeli povezati s sočasno prisotnostjo obeh fenotipov. Najpogostejši sindromi so bili sindromi CATCH 22, CHARGE, Emanuel, Wolf-Hirschhorn, Loeys Dietz tipa 1 in 2, Okamoto, Patau in Say-Barber-Biesecker-Young-Simpson. V vseh spremembah, ki niso bile sekvenčne, smo v literaturi poiskali še vse gene, ki jih povezujemo s hkratno prisotnostjo obeh fenotipov. Takšni geni so bili geni TGFBR1, TGFBR2, CHD7, TBX1, MAPK1, HIC2, HIRA, MEIS2, STAG2, BMP2, PDGFC, PGM1, PGAP3, PTCH1, RPL5, HNRNPK, SORBS2, KAT6B in DGCR2. Congenital abnormalities are considered to be one of the leading causes of neonatal mortality in the modern world of today. The most common congenital abnormalities are orofacial clefts and congenital heart defects. Orofacial cleft is a congenital opening of the lip, palate or mandibular ridge, or it can be a combination of all of them. It is a major esthetic and functional problem as it unites the nasal and oral cavities in various forms and degrees. Congenital heart defect is the result of abnormal development of the heart and/or large blood vessels. It occurs in different segments of the heart and connects them to each other. Despite the high prevalence and importance of orofacial clefts and congenital heart defects, the genetic background of the cause of their co-occurrence is poorly understood. The number of genes involved in their development is actually very large. The aim of this study was to improve the genetic diagnosis of patients with orofacial clefts and congenital heart defects, as only by understanding and identifying the mechanisms of their co-occurrence can we act preventively and treat such cases promptly. Therefore, in this study, we performed a systematic literature review, searching for all reported genetic variants in patients with coexisting orofacial clefts and congenital heart defects. After defining the search profile, we searched the PubMed database for relevant literature. We defined inclusion criteria and then considered those studies that met the criteria. There were 210 such studies, and we identified 611 cases of individuals with more than 150 different genetic variants and coexisting phenotypes. The most frequent genetic variant of all was the 22q11 microdeletion, which was detected 364 times. Other common genetic variants were mutations in the PGM1, MEIS2, STAG2, BMP2, PGAP3 and RPL5 genes, a deletion of 15q14, terminal deletions of 4q and trisomies 22, 10 and 9. Some of the genetic variants also belonged to known syndromes that have already been associated with the co-occurrence of both phenotypes. The most common syndromes were CATCH 22, CHARGE, Emanuel, Wolf-Hirschhorn, Loeys Dietz types 1 and 2, Okamoto, Patau and Say-Barber-Biesecker-Young-Simpson. In all the non-sequence variants, we also searched the literature for all the genes associated with the simultaneous presence of the two phenotypes. Such identefied genes were TGFBR1, TGFBR2, CHD7, TBX1, MAPK1, HIC2, HIRA, MEIS2, STAG2, BMP2, PDGFC, PGM1, PGAP3, PTCH1, RPL5, HNRNPK, SORBS2, KAT6B and DGCR2.

Orofacialne shize (ang. orofacial clefts OFC) so prirojene nepravilnosti ustnice, čeljustnega grebena ali neba oz. kombinacija teh. Posamezniki z OFC imajo pogosto težave z govorom in hranjenjem, poleg tega pa je prisotnost anomalije moteča tudi z estetskega vidika. V Sloveniji se rodi približno 1,67 otroka z OFC na 1.000 rojstev. OFC so posledica kompleksnih genetskih in okoljskih dejavnikov. V primeru t. i. sindromskih shiz, kjer je poleg OFC prisotna še druga simptomatika, je identifikacija vzročnih genov precej uspešna. Nasprotno pa je v primeru t. i. nesindromskih orofacialnih shiz, kjer se uveljavlja teorija o kompleksni etiologiji, z večjim številom medsebojno delujočih genov in sočasnimi vplivi okolja. V preteklosti so opravili več študij, ki so dokazale povezanost posameznih genov z OFC. V okviru magistrske naloge smo želeli proučiti spremembe nukleotidnega zaporedja genov TBX22, IRF6 in GRHL3 ter oceniti njihov vpliv na pojav OFC. V ta namen smo proučevali vzorce DNA petih slovenskih družin, pri katerih je prisoten razcep v orofacialnem področju. V študijo smo vključili tri bolnike s pozitivno družinsko anamnezo in sumom na sindrom Van der Woude ter dva bolnika z nesindromsko obliko shize in brez družinske anamneze. Najprej smo optimizirali pogoje PCR za devet parov oligonukleotidnih začetnikov gena TBX22, nato pa smo v vzorcih posameznikov z OFC pod optimiziranimi pogoji pomnožili izbrane eksone ter blizu ležeča intronska zaporedja genov TBX22, IRF6 in GRHL3 ter produkte PCR poslali na sekvenciranje po Sangerju. Nato smo spremembe, odkrite pri indeksnih bolnikih, tarčno iskali pri njihovih zdravih in obolelih družinskih članih. Skupno smo odkrili 12 genetskih sprememb šest v genu GRHL3 (rs79338443, rs2486668, rs45511995, rs11576645, rs41268753 in rs545809), pet v genu IRF6 (rs121434229, rs7552506, rs2013162, rs2235375 in rs1553247595) in eno v genu TBX22 (rs1057515991). Glede na patogeni potencial smo odkrili eno »patogeno« in eno »verjetno patogeno« spremembo, na podlagi katerih lahko v treh družinah potrdimo sum na sindrom Van der Woude. Poleg tega smo odkrili eno spremembo »negotovega pomena«, preostalih devet pa je »benignih«. Glede na vrsto spremembe so med odkritimi genetskimi spremembami štiri drugačnosmiselne, dve sinonimni, ena nesmiselna, ena sprememba 3' UTR regije in štiri intronske spremembe, med katerimi je ena sprememba spojitvenega mesta. Orofacial clefts (OFC) are congenital abnormalities of the lip, alveolus, or palate or a combination of those. Individuals with OFC often face difficulties with eating and speeking. Apart from that the anomaly also represents an aesthetic issue. In Slovenia, about 1,67 children are born with OFC per 1000 births. OFC are caused by complex genetic and environmental factors. In the case of the syndromic orofacial clefts, the identification of the causal genes is fairly successful. On the other hand, it is not so in the case of non-syndromic orofacial clefts, where a theory of complex etiology and multiple gene factoring with simultaneous environmental influences was established. Multiple studies in the past have shown a correlation of certain genes with OFC. The goal of this master’s thesis was to study the changes in the nucleotide sequence of the genes TBX22, IRF6 and GRHL3, and assess their influence on the incidence of OFC. For this purpose, DNA samples of 5 Slovenian families with the incidence of OFC were studied. 3 patients with OFC, positive familial history and suspected Van der Woude syndrome, and 2 patients with a non-syndromic OFC and no family history of OFC were included in the study. First, PCR conditions for nine pairs of oligonucleotide primers of the gene TBX22 were optimized. Next, choosen exons and nearby lying intron sequences of the genes TBX22, IRF6 and GRHL3 of the individuals with OFC were multiplied, under previously optimized PCR conditions. PCR products were sequenced with the Sanger sequencing method. Discovered changes in the nucleotide sequence of index cases were then looked for in their family members with and without OFC. Overall, we discovered 12 genetic variants six in the gene GRHL3 (rs79338443, rs2486668, rs45511995, rs11576645, rs41268753 and rs545809), five in the gene IRF6 (rs121434229, rs7552506, rs2013162, rs2235375 and rs1553247595), and one in the gene TBX22 (rs1057515991). Looking at the pathogenic potential, one »pathogenic« and one »likely pathogenic« sequence variant were found that can confirm the suspicion of the Van der Woude syndrome in three families. Furthermore, one »variant of uncertain significance« and nine »benign« variants were found. According to the type of the variants, the following genetic variants were found: four mis-sense variants, two synonymous variants, one non-sense variant, one variant in the 3' UTR region, and four intronic variants, one of them being the splice site variant.

Human genetic variants occur throughout the whole genome, both in the coding and non-coding regions. In addition to defining genetic diversity among individuals, they may, in some instances, cause various genetic diseases, many of which have their onset in childhood. In the first part of the review article, we describe different types of genetic variants, ranging from minor sequence changes to major chromosomal aberrations. This is followed by a presentation of some of the most important approaches (cytogenetic and molecular genetic) that are used to detect genetic variants in clinical practice. We put greater emphasis on sequencing methods, as they allowed us to gain insight into the nucleotide sequence of the entire genome, and therefore revolutionised the field of molecular genetic diagnostics. We also highlight the problems that arise in determining genetic variants through next-generation sequencing (NGS) methods and outline the benefits of the latest sequencing techniques − long-read sequencing. Today, a substantial proportion of human genetic variants associated with the development of genetic diseases, are still unknown. Therefore, we conclude the article by presenting some possible approaches that could improve the detection of genetic variants at the individual level.

Človeške papilomaviruse (HPV) etiološko povezujemo z nastankom številnih benignih in malignih sprememb kože in sluznic pri ljudeh. Po podatkih Mednarodnega referenčnega centra za HPV je bilo do novembra 2017 uradno priznanih 206 genotipov HPV, nedavno pa so dokazali, da naj bi obstajalo še vsaj 400 potencialno novih, še neopredeljenih genotipov HPV. Naša raziskovalna skupina je leta 2014 v vzorcu analnega brisa dokazala prisotnost novega genotipa HPV, HPV-204, ki smo ga uvrstili v rod Mupapillomavirus (Mu-PV), kamor sta bila do tedaj uvrščena le dva genotipa HPV (HPV-1 in HPV-63). V doktorski nalogi smo želeli natančno filogenetsko in molekularno opredeliti HPV-204. Poleg tega smo na podlagi testiranja reprezentativne zbirke kliničnih vzorcev želeli opredeliti tkivni tropizem in klinični pomen novega genotipa HPV-204 ter sorodnih genotipov HPV-1 in HPV-63. V zadnjem delu doktorske naloge smo želeli opredeliti genetsko raznolikost genotipov iz rodu Mu-PV s pomnoževanjem in neposrednim določanjem nukleotidnega zaporedja nekodirajočega genomskega področja (LCR). S pomočjo več programov in prosto dostopnih spletnih aplikacij smo v prvem delu doktorske naloge opredelili značilna genomska področja celotnega virusnega genoma HPV-204. Celotno nukleotidno zaporedje HPV-204 je sestavljeno iz 7.227 baznih parov (bp) s 37,8% deležem bp gvanin-citozin. HPV-204 izkazuje organizacijo genoma, ki je značilna za predstavnike rodu Mu-PV, saj vsebuje zapis za šest zgodnjih (E6, E7, E1, E2, E8 in E4) in dve pozni (L1 in L2) virusni beljakovini, vendar nima zapisa za zgodnjo beljakovino E5. Na podlagi filogenetske analize gena L1 vseh uradno priznanih genotipov HPV smo potrdili preliminarno uvrstitev HPV-204 v rod Mu-PV, in sicer v novo vrsto Mu-3, pri čemer lahko sklepamo, da je HPV-204 najbolj soroden s HPV63, s katerim izkazuje 66,9% skladnost v genu L1, medtem ko s HPV-1 izkazuje 66,1% skladnost v genu L1. Tkivni tropizem in klinični pomen okužb z genotipi iz rodu Mu-PV smo opredelili s testiranjem reprezentativne zbirke kliničnih vzorcev (n=1.006), za kar smo razvili in uvedli analitično zelo občutljive kvantitativne HPV-204, HPV-1 in HPV-63 tipsko-značilne reakcije pomnoževanja s polimerazo v realnem času (RT-PCR). Odkrili smo 11 neodvisnih izolatov HPV-204, in sicer v brisu materničnega vratu (1/116 0,8%), vzorcu kožne bradavice (1/43 2,3%), brisih analnega kanala (4/110 3,6%) in brisih penisa (5/110 4,5%). V sklopu doktorske naloge smo skupno odkrili 27 neodvisnih izolatov HPV-1, ki smo jih odkrili v brisih nosnega dela žrela (2/110 1,8%), dlačnem mešičku obrvi (1/110 0,9%), vzorcih kožnih bradavic (21/43 48,8%) in vzorcih anogenitalnih bradavic (3/40 7,5%), ter 19 neodvisnih izolatov HPV-63, ki smo jih odkrili v brisih nosnega dela žrela (3/110 2,7%), brisih ustne votline (2/105 1,9%), dlačnih mešičkih obrvi (4/110 3,6%), vzorcih kožnih bradavic (9/43 20,9%) in brisu analnega kanala (1/110 0,9%). Na podlagi testiranja arhivskih vzorcev benignih in malignih sprememb ter klinično nespremenjene kože in sluznic smo dokazali, da vsi genotipi iz rodu Mu-PV izkazujejo dvojni tkivni tropizem. V vzorcu kožne bradavice, v katerem smo dokazali HPV-204, je bilo virusno breme HPV-204 izjemno nizko (7,4 x 10-7 virusnih kopij/celico), kar nakazuje na latentno, klinično nepomembno okužbo. Čeprav je bilo virusno breme HPV-1 večinoma nizko (razpon 5,9 x 10-6 – 1,8 x 10-2 virusnih kopij/celico), je bil HPV-1 najverjetnejši povzročitelj treh kožnih bradavic, v katerih smo dokazali visoko virusno breme HPV-1 (razpon 8,5 x 103 – 5,2 x 104 virusnih kopij/celico). Virusno breme HPV-63 je bilo podobno kot pri HPV-1 v večini vzorcev nizko (razpon 3,2 x 10-6 – 1,1 x 10-2 virusnih kopij/celico), HPV-63 pa je predstavljal najverjetnejšega povzročitelja le v vzorcu kožne bradavice z visokim virusnim bremenom (3,9 x 102 virusnih kopij/reakcijo). Z opredelitvijo virusnega bremena HPV-204, HPV-1 in HPV-63 smo potrdili, da je okužba s temi genotipi HPV večinoma klinično neaktivna, pri čemer sta HPV-1 in HPV-63 etiološka povzročitelja zgolj posameznih kožnih bradavic, medtem ko HPV-204 nismo uspeli povezati z nastankom kožnih bradavic. V zadnjem delu doktorske naloge smo na osnovi analize nukleotidnega zaporedja nekodirajočega genomskega področja LCR genotipov iz rodu Mu-PV odkrili eno podtipsko različico HPV-204, ki je vsebovala eno točkasto mutacijo, šest podtipskih različic HPV-1, ki so vsebovale 1-19 nukleotidnih zamenjav oziroma izpadov, ter dve podtipski različici HPV-63, pri čemer je prva podtipska različica vsebovala 13, druga pa šest nukleotidnih zamenjav oziroma izpadov. Predvidevamo, da opredeljene podtipske različice posameznih genotipov HPV niso povezane z značilno klinično sliko ali anatomskim področjem okužbe. Izsledke raziskave, ki so predstavljeni v doktorski nalogi, smo objavili kot znanstveni raziskovalni članek v reviji, ki jo citira Science Citation Index (SCI) (IF2016=2,806): Šterbenc A, Hošnjak L, Chouhy D, Bolatti EM, Oštrbenk A, Seme K, Kocjan BJ, Luzar B, Giri AA, Poljak M. Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63. PLoS One 2017 12:e0175892. Human papillomaviruses (HPV) are etiologically linked with the development of various benign and malignant neoplasms in humans. As of November 2017, a total of 206 HPV types were officially recognized by the HPV Reference center at the Karolinska Institute in Sweden, although it has recently been shown that there are still at least 400 novel HPV types that have not yet been identified. In 2014, our research group identified a novel HPV, HPV-204, in an anal canal swab sample. HPV-204 was preliminarily classified into the genus Mupapillomavirus (Mu-PV) that consisted of only two HPV types: HPV-1 and HPV-63. In this study, our aim was to perform comprehensive phylogenetic and molecular characterization of HPV-204. In the second part of the study, we wanted to determine the tissue tropism and potential clinical significance of Mu-PVs by testing a representative collection of clinical specimens. Finally, we wanted to evaluate the genetic variability of Mu-PVs by amplifying and sequencing the long control region (LCR) of each Mu-PV. Using several programs and freely available online applications, we determined characteristic genomic regions of HPV-204. The 7,227 base pair (bp) long genome of HPV-204 with a GC ratio of 37.8% exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1), while lacking the E5 ORF. Based on phylogenetic analysis of the L1 gene of all officially recognized HPV types, HPV-204 clusters within the Mu-PV genus, species Mu-3. HPV-204 is most similar to HPV-63, since the L1 gene of HPV-204 exhibits 66.9% nucleotide sequence identity with the L1 gene of HPV-63 and 66.1% with the L1 gene of HPV-1. To evaluate the tissue tropism and potential clinical relevance of Mu-PVs, we tested a representative collection of clinical samples (n=1,006) using three newly developed and highly sensitive quantitative type-specific real-time polymerase chain reactions (RT-PCRs) for detection of HPV-204, HPV-1, and HPV-63. A total of 11, 27, and 19 HPV-204, HPV-1, and HPV-63 isolates were obtained, respectively. HPV204 was detected in cervical swabs (1/116 0.8%), cutaneous wart (1/43 2.3%), swabs of the anal canal (4/110 3.6%), and swabs of the penile surface (5/110 4.5%). HPV-1 was detected in nasopharyngeal swabs (2/110 1.8%), eyebrow hair follicle (1/110 0.9%), cutaneous warts (21/43 48.8%), and anogenital warts (3/40 7.5%), while HPV-63 was detected in nasopharyngeal swabs (3/110 2.7%), swabs of the oral cavity (2/105 1.9%), eyebrow hair follicles (4/110 3.6%), cutaneous warts (9/43 20.9%), and swab of the anal canal (1/110 0.9%). We have confirmed that all Mu-PVs exhibit dual tissue tropism, since HPV-204, HPV-1, and HPV-63 were detected in cutaneous, mucosal, and mucocutaneous epithelia. HPV-204 viral load was extremely low in a single HPV-204-positive cutaneous wart (7.4 × 10&#8722 7 viral copies/cell). Hence, etiological association between infection with HPV-204 and the development of cutaneous warts could not be confirmed. Although the majority of HPV-1-positive samples contained low number of viral copies (range 5.9 x 10-6 – 1.8 x 10-2 viral copies/cell), HPV-1 was the most likely causative agent of three cutaneous warts with high HPV-1 viral load (range 8.5 x 103 – 5.2 x 104 viral copies/cell). Similarly, HPV-63 viral load was low in the majority of HPV-63-positive tissue samples (range 3.2 x 10-6 – 1.1 x 10-2 viral copies/cell). However, HPV-63 was the most likely causative agent of one common wart, where it was present in high viral copy numbers (3.9 x 102 viral copies/cell). Thus, we have confirmed that the majority of infections with Mu-PVs are clinically latent, whereas HPV-1 and HPV-63 may be the causative agents of sporadic cases of common warts. In contrast, we were not able to establish a causative association between the presence of HPV-204 and the development of common warts. Finally, we evaluated the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV-204, HPV-1, and HPV-63. We identified one genetic variant of HPV-204 that contained a single nucleotide polymorphism (SNP), six genetic variants of HPV-1 that contained 1-19 SNPs and/or nucleotide deletions and two genetic variants of HPV-63 that contained 6-13 SNPs and/or nucleotide deletions. Based on the obtained data, none of the genetic variants could be reliably associated with specific clinical manifestations or anatomical location of infection. Part of the doctoral thesis was published as a scientific paper in a journal cited by the Science Citation Index (SCI) (IF2016=2.806): Šterbenc A, Hošnjak L, Chouhy D, Bolatti EM, Oštrbenk A, Seme K, Kocjan BJ, Luzar B, Giri AA, Poljak M. Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63. PLoS One 2017 12:e0175892.