Opredelitev genetskih vzrokov prezgodnje pubertete : magistrski študijski program Laboratorijska biomedicina

Mivšek, Kaja (author), Trebušak Podkrajšek, Katarina (mentor), Avbelj Stefanija, Magdalena (co-mentor)

Interakcije med genetskimi in okoljskimi dejavniki določajo čas začetka spolnega dozorevanja. Puberteta se prične, ko se hipotalamično-hipofizno-gonadna os ponovno aktivira. Takrat opazimo razvoj žleznega tkiva dojk pri deklicah oz. povečanje mod pri dečkih ter sočasno pospešeno rast in dozorevanje kosti. Kadar se omenjeni znaki pojavijo pred pričakovano starostjo, je možen vzrok zanje centralna prezgodnja puberteta. Prezgodnji spolni razvoj pomeni premik rodne dobe, ki ga povezujemo s tveganjem za razvoj nekaterih bolezni, večjo nagnjenostjo k nižji odrasli postavi in možnostjo nastanka psihosocialnih motenj. Za opredelitev morebitnega dednega vzroka bolezni so potrebne molekularne genetske preiskave. Namen magistrske naloge je bil odkriti genetske spremembe, ki bi lahko bile odgovorne za prezgodnjo puberteto. Določili smo nukleotidno zaporedje celotnega genoma štirih družin s prezgodnjo puberteto, ki se deduje po materi. Uporabili smo pristop sekvenciranja celotnega genoma in z bioinformatskoo analizo skušali opredeliti možne genetske vzroke za prezgodnjo puberteto. Z uporabo orodij in algoritmov smo našli spremembe, ki so pri vseh štirih družinah ležale v istem genu ZNF717 in jih tako prepoznali kot možno vzročne. Kvanitativno smo ocenili patogenost omenjenih sprememb. Pri članih četrte družine smo prisotnost najverjetnejše patogene spremembe preverjali s potrditvenim sekvenciranjem po Sangerju. Rezultat potrditvene analize je pokazal odsotnost iskane spremembe, kar kaže na napako v pristopu sekvenciranja celotnega genoma oziroma sledeče bioinformatske analize. Razpravljali smo o vzrokih za lažno pozitiven rezultat ter predstavili slabosti pristopa s sekvenciranjem celotnega genoma in bioinformatske analize. Rezultati prvotnih analiz niso bili zanesljivi, kar pomeni, da potencialno vzročne genetske spremembe pri analiziranih družinah s prezgodnjo puberteto ostajajo neopredeljene. Naši rezultati kažejo, da je preverjanje rezultatov sekvenciranja naslednje generacije s potrditveno metodo nujno potrebno za zanesljivo genetsko diagnostiko.

• Language: Slovenian
• Keywords: prezgodnja puberteta sekvenciranje genoma bioinformatska analiza sekvenciranje po Sangerju lažno pozitiven rezultat
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [K. Mivšek]
• Year: 2018
• Number of pages: IV, 62 str.
• PID: 20.500.12556/RUL-120263
• UDC: 616-074:612.661(043.3)
• COBISS.SI-ID: 4462705
• Publication date in RUL: 17.09.2020

Secondary language

• Language: English
• Title: The identification of the genetic cause of precocious puberty

Abstract

Interactions between genetic and environmental factors determine the timing of sexual maturation. Puberty begins when the hypothalamic-pituitary-gonadal axis is reactivated. At that time, mammary gland in girls and testes in boys are developed and growth and bone maturation are accelerated. When this pubertal characteristics occur before the expected age, possible cause for them is central precocious puberty. Premature pubertal development results in the shift of a fertile period, which is associated with the risk of developing certain diseases, a greater tendency towards the lower final adult stature and increased potential for psychosociological disorders. Molecular genetic testing is needed to identify the potential hereditary cause of the disease. The aim of this study was to detect genetic variants, that could be responsible for precocious puberty. We determined the sequence of the entire genome in members of four families with maternally inherited precocious puberty. We applied the whole genome sequencing followed by bioinformatic approaches in order to identify the possible genetic causes of precocious puberty. Using tools and algorithms, we identified variants in all four families located in the same gene ZNF717 and therefore determined them as potentially pathogenic. We quantitatively assessed the pathogenicity of these variants. In members of the fourth family, we verified the presence of the most likely pathogenic variant by confirmative Sanger sequencing. Results of Sanger analysis showed, that the desired variant was not present, indicating error in the approach of whole genome sequencing and subsequent bioinformatic analysis. The causes of the false positive result were discussed and the weaknesses of the whole genome sequencing method and the bioinformatic analysis were presented. The results of the initial analyses were not reliable, therefore potentially causative genetic variants in analyzed families with precocious puberty remain undetermined. Our results showed that verification of the results of next generation sequencing with a secondary method is necessary for reliable genetic diagnostics.

• Keywords: familial central precocious puberty sequencing of the entire genome bioinformatics analysis Sanger sequencing a false positive result