Analiza genetskih in metilacijskih sprememb gena MKRN3 pri bolnikih s prezgodnjo puberteto

Novak, Eva (author), Trebušak Podkrajšek, Katarina (mentor), Avbelj Stefanija, Magdalena (comentor)

O prezgodnji puberteti govorimo, kadar se znaki pubertetnega razvoja pojavijo pred pričakovano starostjo. Pri dečkih se poveča velikost mod, deklicam se pričnejo razvijati prsi, kosti pa pospešeno rastejo in dozorevajo. Nastop pubertete je pod velikim vplivom socialnih in okoljskih dejavnikov, na področju centralne prezgodnje pubertete pa sta genetika in epigenetika predmet intenzivnega raziskovanja. Gre za obliko prezgodnje pubertete, ki nastane zaradi prezgodnje aktivacije hipotalamično-hipofizno-gonadne osi. V približno četrtini primerov centralne prezgodnje pubertete govorimo o družinski prezgodnji puberteti. Uporaba molekularne genetike je zato pomembna v procesu določitve morebitnega genetskega vzroka bolezni in za potrebe genetskega testiranja družinskih članov bolnikov. Namen magistrske naloge je bil v populaciji sporadičnih bolnikov in bolnikov z družinsko centralno prezgodnjo puberteto ter njihovih družinskih članih opredeliti vzročne spremembe v genu MKRN3. Nukleotidno zaporedje kodirajoče regije gena smo določili s sekvenciranjem po Sangerju. Pri bolnikih, ki s sekvenciranjem niso imeli opredeljenih vzročnih sprememb, družinska anamneza pa je nakazovala na paternalno dedovano centralno prezgodnjo puberteto, smo z metodo MS-MLPA iskali spremembe v številu kopij in metilacijskem vzorcu gena MKRN3 in DLK1. S sekvenciranjem po Sangerju smo v dveh rodovnikih z družinsko centralno prezgodnjo puberteto odkrili in potrdili predhodno že opisano vzročno spremembo, v enem rodovniku pa smo opredelili potencialno novo kandidatno varianto. Z in silico napovednimi algoritmi in segregacijsko analizo smo dokazali patološkost nove variante. Pri preiskovancih s sporadično obliko bolezni nismo odkrili vzročnih sprememb v kodirajoči regiji gena MKRN3. Določili nismo nobenih delecij/duplikacij in metilacijskih nepravilnosti v genih MKRN3 in DLK1, zato smo razpravljali o omejitvah MS-MLPA metode. S sekvenciranjem po Sangerju je mogoče opredeliti vzročne spremembe v genu MKRN3. Z analizo smo razširili spekter variant, povezanih s centralno prezgodnjo puberteto, v prihodnosti pa bo ključna identifikacija novih tarčnih genov reproduktivne osi.

• Language: Slovenian
• Keywords: Družinska centralna prezgodnja puberteta, sekvenciranje po Sangerju, MS-MLPA, MKRN3, nova kandidatna sprememba
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107976
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Analysis of the MKRN3 gene variants and aberrant methylation in patients with precocious puberty

Abstract

Precocious puberty is defined as the development of pubertal signs before the estimated age. It is characterised by testicular enlargement in boys, breast development in girls and bone growth acceleration. The onset of puberty is under the great influence of social and environmental factors; however, genetics and epigenetics are two fields of intensive research in central precocious puberty. This is a form of precocious puberty caused by premature activation of hypothalamic-pituitary-gonadal axis. In approximately quarter of cases the cause is genetic and that is why the application of molecular genetic assays is vital in the process of determining potential hereditary cause of the disease and for genetic counselling in patient’s relatives. The aim of our study was to evaluate the presence of causative genetic variants in the MKRN3 gene in a group of sporadic patients, patients with familial central precocious puberty and their relatives. Nucleotide sequence of coding region was analysed using Sanger sequencing method. Patients with no identified genetic variants in target gene and family history consistent with paternal manner of inheritance were further analysed for copy number variations and changes in methylation pattern in genes MKRN3 and DLK1 using methylation-specific MLPA method. We identified previously reported causative genetic variant in two pedigrees with familial central precocious puberty and novel potential candidate variant in another pedigree. The results of in silico predictive programs and segregation analysis are supporting evidence of pathogenic impact of this novel variant. We defined no genetic changes in coding sequence of the MKRN3 gene in sporadic patients. No large deletions/duplications and aberrant methylation changes were detected using methylation-specific MLPA assay. Therefore, the limitations of this method were discussed. Sanger sequencing can be used for defining genetic variants in the MKRN3 gene. In our study we expanded the spectrum of variants implicated in central precocious puberty. Apart from this, identification of new target genes in reproductive axis will be of great importance.

• Keywords: Familial central precocious puberty, Sanger sequencing, methylation-specific MLPA method, MKRN3, novel variant