Proučevanje sprememb nukleotidnega zaporedja genov TBX22, IRF6 in GRHL3 v družinah z orofacialnimi shizami

Bokal, Meta (avtor), Karas Kuželički, Nataša (mentor), Slavec, Lara (komentor)

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.

• Jezik: Slovenski jezik
• Ključne besede: genetske spremembe, nesindromske orofacialne shize, sindrom Van der Woude, študija družin
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-137923
• Datum objave v RUL: 06.07.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: A study of nucleotide sequence changes in TBX22, IRF6 and GRHL3 genes in families with orofacial clefts

Izvleček

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.

• Ključne besede: genetic variants, non-syndromic orofacial clefts, Van der Woude syndrome, family study