20.500.12556/RUL-186942 Detekcija različic v genu SLITRK6 in njihov potencialni vpliv na glikozilacijsko stanje proteinskega produkta Detection of variants in the SLITRK6 gene and their potential impact on the glycosylation state of the protein product Protein SLITRK6 (SLIT in NTRK podobni protein 6) je monomeren transmembranski protein iz družine SLITRK, ki je povezan z delovanjem živčnega sistema. Za proteine družine SLITRK so značilne z levcinom bogate regije. SLITRK6 je eden izmed ključnih dejavnikov pri sinaptogenezi in je posledično selektivno izražen v centralnem živčnem sistemu. V literaturi je SLITRK6 povezan tudi s kancerogenezo. Opaženo je bilo zvišano izražanje mRNA SLITRK6 pri raku debelega črevesa in danke, podobno velja pri raku sečnega mehurja, kjer so opazili zvišano izražanje tako na mRNA kot na proteinski ravni. Pri hepatoceličnem karcinomu so potrdili, da SLITRK6 deluje kot spodbujevalec kancerogeneze, njegova funkcija pa je pogojena s pridobljenimi nukleotidnimi različicami v genu. Prav tako obstaja povezava med nastankom adenokarcinoma pljuč in SLITRK6. Na osnovi deglikozilacije (neobjavljeni rezultati) smo ugotovili, da je protein SLITRK6 glikoziliran. Namen dela je bil ugotoviti, ali ima glikozilacija proteina SLITRK6 funkcionalno vlogo in ali je glikozilacijski vzorec pogojen z različicami, pogostimi pri raku. Z več programi smo napovedali mesta N- in O-glikozilacije na aminokislinskem zaporedju SLITRK6 in jih vrednostno opredelili glede na kakovost strukture modela proteina. S sekvenciranjem po Sangerjevi metodi smo določili zaporedje v genu SLITRK6 pri 42 bolnikih z rakom debelega črevesa in danke in 8 bolnikih z rakom prostate. DNA smo izolirali iz tkivnih vzorcev, fiksiranih v formalinu in vklopljenih v parafin. Opredelili smo 21 možnih različic, ki bi lahko vplivale tako na strukturo kot tudi na glikozilacijo SLITRK6. Z uporabo programov za poravnavo, identifikacijo, oceno patogenosti in podatkovnih zbirk Ensembl, COSMIC, ClinVar in TCGA smo opredelili 10 potencialnih drugačnosmiselnih različic, 3 so imele največji napovedani škodljivi učinek: c.1670T>A, c.665G>T in c.485T>C. Preverili smo njihov vpliv na strukturo in glikozilacijski vzorec. Potrdili smo različico c.665G>T, ki je že bila zaznana pri adenokarcinomu pljuč, in jo vnesli v podatkovno zbirko COSMIC. Neposrednega vpliva opredeljenih različic na glikozilacijo nismo potrdili, modeliranje pa je pokazalo, da ob prisotnosti različic pride do več primerov steričnega oviranja v proteinski strukturi in do vzpostavitve stop kodona. V podatkovni zbirki COSMIC smo identificirali potrjene mutacije pri drugih rakih, ki bi lahko vplivale na glikozilacijsko stanje SLITRK6. SLITRK6 (SLIT and NTRK-like protein 6) is a monomeric transmembrane protein belonging to the SLITRK family, members of which are characterized by leucine-rich repeat (LRR) domains and are predominantly associated with nervous system function. SLITRK6 plays a key role in synaptogenesis and is primarily expressed in the central nervous system. In addition to its neurological role, SLITRK6 has been implicated in carcinogenesis. Elevated mRNA expression has been reported in colorectal cancer, and a similar upregulation at both the mRNA and protein levels has been observed in bladder cancer. In hepatocellular carcinoma, SLITRK6 has been shown to promote carcinogenesis, with its function modulated by acquired nucleotide variants. An association between SLITRK6 expression and the development of lung adenocarcinoma has also been described. Based on unpublished deglycosylation data, we established that SLITRK6 is a glycoprotein. This prompted us to investigate whether glycosylation plays a functional role in SLITRK6- associated carcinogenesis, and whether the glycosylation pattern is influenced by variants frequently occurring in cancer tissue. Potential N- and O-glycosylation sites were predicted across the SLITRK6 amino acid sequence using multiple tools and further evaluated based on the local quality of the protein structural model. Using Sanger sequencing, we determined SLITRK6 sequences from formalin-fixed, paraffin-embedded (FFPE) tissue samples from 42 colorectal cancer patients and 8 prostate cancer patients. A total of 21 candidate variants potentially affecting SLITRK6 structure and glycosylation were identified. Subsequent analysis using alignment and pathogenicity prediction tools, together with cross-referencing against the Ensembl, COSMIC, ClinVar, and TCGA databases, yielded 10 candidate missense variants, three of which were predicted to be pathogenic: c.1670T>A, c.665G>T, and c.485T>C. The impact of these variants on protein structure and glycosylation patterns was assessed. Although no direct effect on glycosylation was confirmed, analysis of the SLITRK6 model revealed that several variants introduce steric clashes within the protein structure or lead to the formation of a premature stop codon. Variant c.665G>T, previously reported in lung adenocarcinoma tissue, was validated and subsequently submitted to the COSMIC database. Additionally, we identified variants in other cancer types from the COSMIC database that may potentially influence the glycosylation status of SLITRK6. SLITRK6 kancerogeneza različice glikozilacija SLITRK6 carcinogenesis variants glycosylation true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-07 13:10:01 2026-09-07 13:10:13 2026-09-24 11:20:59 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25772 292344323 Tomsic_Jakob_-_Detekcija_razlicic_v_genu_SLITRK6_in_njihov_potencialni_vpliv_na_glikozilacijsko_.pdf Tomsic_Jakob_-_Detekcija_razlicic_v_genu_SLITRK6_in_njihov_potencialni_vpliv_na_glikozilacijsko_.pdf 1 232F6D48AF7E443D5ACFBDBFB4D53795 bd1ed10ba1d7c578d00153229a98f8abac49d801224cb0e5b2e163931f092720 b55e1a2e-aaac-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246342 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0