Bioinformatična analiza rezultatov sekvenciranja naslednje generacije in analiza segregacije sprememb v genih BMPR1B, COL11A1 in MCPH1 v družinah z orofacialno shizo

Potočnik, Aljaž (avtor), Karas Kuželički, Nataša (mentor), Urbančič, Dunja (komentor)

Orofacialne shize (OFC) so ena najpogostejših prirojenih nepravilnosti obraza na svetu, ki se lahko klinično izrazijo kot razcep ustnice, čeljustnega grebena in/ali neba ter imajo tudi genetsko predispozicijo. Ločimo nesindromske oblike, za katere so značilni izolirani razcepi ustnice in/ali neba brez pridruženih drugih anomalij obraza in/ali drugih organskih sistemov, ter sindromske oblike, ki so redkejše in pri katerih se poleg shize pojavljajo še druge razvojne nepravilnosti. Predstavljajo velik javnozdravstveni problem zaradi relativno visoke prevalence, dolgotrajnega zdravljenja in vseh posledic, ki jih povzročajo prizadetim posameznikom in njihovim družinam. Namen magistrske naloge je bil identifikacija genetskih sprememb, ki bi lahko pojasnile pojav OFC. Na Univerzi v Ljubljani, Fakulteti za farmacijo so v okviru študije iskanja genetskih dejavnikov tveganja za OFC izvedli sekvenciranje celotnega eksoma 28 indeksnih bolnikov iz 25 družin z družinsko anamnezo OFC, pri katerih do sedaj še niso odkrili vzroka anomalije. V okviru magistrske naloge smo pri indeksinh bolnikih pregledali 30 kandidatnih genov na prisotnost potencialno patogenih genetskih sprememb, določenih s sekvenciranjem celotnega eksoma. S pomočjo bioinformatičnih orodij smo analizirali sledeče kandidatne gene: BANF1, BBIP1, BBS1, BBS10, BBS12, BBS2, BBS4, BBS5, BBS7, BBS9, BCOR, BCS1L, BEST, BLM, BMP1, BMPER, BMPR1A, BMPR1B, BRAF, BUB1B, C12orf57, C15orf41, C5orf42, C6, CACNA1C, CAMK2G, CANT1, CAPN5, CASK, CASP7. Z modeli in silico smo želeli določiti, ali je katera od genetskih sprememb prepoznana kot verjetno patogena ali patogena po klasifikaciji Ameriškega kolegija za medicinsko genetiko in genomiko. Pri dveh bolnikih smo odkrili eno verjetno patogeno spremembo v genu BMPR1B, ki smo jo v nadaljevanju potrjvali s sekvenciranjem po Sangerju pri obeh bolnikih s to spremembo in njunih zdravih in prizadetih sorodnikih. Dodatno smo potrjevali še tri spremembe v genih COL11A1 in MCPH1, ki so bile kot verjetno patogene ali patogene že predhodno identificirane z bioinformatičnimi orodji v okviru te študije. Pri družini OFC_059 smo potrdili, da drugačnosmiselna sprememba v genu COL11A1 segregira z bolezenskim fenotipom, kar nakazuje na njeno verjetno vlogo pri pojavu OFC v tej družini. V preostalih družinah povezave nismo uspeli enoznačno dokazati. V nekaterih primerih spremembe niso bile zaznane ali niso dosledno segregirale z bolezenskim fenotipom, v enem primeru pa je bila možna razlaga z nepopolno penetranco, kar zmanjšuje verjetnost, da bi obravnavane variante predstavljale glavni vzročni dejavnik OFC v teh družinah.

• Jezik: Slovenski jezik
• Ključne besede: orofacialne shize, genetske spremembe, bioinformatična analiza, analiza segregacije, študija družin
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-181866
• Datum objave v RUL: 17.04.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Bioinformatic analysis of next-generation sequencing results and segregation analysis of variants in the BMPR1B, COL11A1 and MCPH1 genes in families with orofacial cleft

Izvleček

Orofacial clefts (OFC) are one of the most common congenital facial abnormalities in the world, which can clinically manifest as a cleft lip, alveolus, and/or palate, and also have a genetic predisposition. We distinguish between non-syndromic forms, which are characterized by isolated facial celfts without other facial anomalies or defects of other organs, and syndromic forms, which are rarer and in which other developmental abnormalities occur in addition to the cleft. They represent a major public health problem due to their relatively high prevalence, long-term treatment, and all the consequences they cause for affected individuals and their families. The aim of the master's thesis was to identify genetic variants that could explain the occurrence of OFC. At the University of Ljubljana, Faculty of Pharmacy, as part of a study of genetic factors of OFC, whole exome sequencing was performed on 28 index patients from 25 families with a family history of OFC in whom the cause of the anomaly had not yet been discovered. As part of a master's thesis, we reviewed 30 candidate genes in index patients for potentially pathogenic genetic variants identified by whole exome sequencing. Using bioinformatic tools, we analyzed the following candidate genes: BANF1, BBIP1, BBS1, BBS10, BBS12, BBS2, BBS4, BBS5, BBS7, BBS9, BCOR, BCS1L, BEST, BLM, BMP1, BMPER, BMPR1A, BMPR1B, BRAF, BUB1B, C12orf57, C15orf41, C5orf42, C6, CACNA1C, CAMK2G, CANT1, CAPN5, CASK, CASP7. Using in silico models, we wanted to determine whether any of the genetic variants were identified as likely pathogenic or pathogenic according to the classification of the American college of medical genetics and genomics. In two patients, we discovered one likely pathogenic variant in the BMPR1B gene, which we subsequently confirmed by Sanger sequencing in both patients with this variant and their healthy and affected relatives. We additionally confirmed three variants in the COL11A1 and MCPH1 genes, which had already been identified as likely pathogenic or pathogenic using bioinformatic tools in this study. In the OFC_059 family, we confirmed that a missense mutation in the COL11A1 gene segregates with the disease phenotype, indicating its probable role in the occurrence of OFC in this family. In the other families, we were unable to demonstrate a clear association. In some cases, the changes were not confirmed or did not consistently segregate with the disease phenotype, and in one case, incomplete penetrance was a possible explanation, which reduces the likelihood that the variants in question are the main causative factor of OFC in these families.

• Ključne besede: orofacial clefts, genetic changes, bioinformatic analysis, segregation analysis, family-based study