Opredelitev genetskih sprememb pri bolnikih z von Willebrandovo boleznijo tipa 2

Globokar, Barbara (author), Trebušak Podkrajšek, Katarina (mentor), DEBELJAK, MARUŠA (co-mentor)

Von Willebrandova bolezen (VWB), ki je posledica genetskih sprememb na genu VWF, je najpogostejša podedovana motnja v strjevanju krvi. Molekularno genetska analiza gena VWF je zelo kompleksna in se v splošni laboratorijski praksi v Sloveniji še ne uporablja. Razlog je visoka polimorfnost gena VWF in njegovega psevdogena (VWFP1), ki moti molekularno genetske analize, tudi sekvenciranje po Sangerju in sekvenciranje naslednje generacije (NGS). Namen magistrske naloge je opredeliti genetske vzroke VWB tipa 2 pri slovenskih bolnikih. V raziskavo smo vključili 26 bolnikov pri katerih obstaja sum na VWB tipa 2. Uvedli smo pristop za specifično pomnoževanje eksona 28 gena VWF, ne pa psevdogena VWFP1. Pomnoženim odsekom smo nato določili nukleotidno zaporedje s sekvenciranjem po Sangerju. Spremembe smo glede na ACMG klasifikacijo opredelili kot patološke, verjetno patološke, spremembe z neznanim pomenom (VUS) in benigne. Vse, razen benignih sprememb, smo podrobno opisali in določili njihov klinični pomen. Na koncu smo spremembe pri bolnikih primerjali s fenotipom bolnikov, opisanih v drugih populacijah. Frekvenca VWB tipa 2A v preiskovani skupini bolnikov s sumom na VWB tipa 2 znaša 25 % in za tip 2B 12,5 %. Pri bolnikih nismo opredelili sprememb, ki bi bile lahko vzrok za VWB tipa 2M in 2N. V primerjavi z drugimi populacijami je v Sloveniji večji delež VWB tipa 2A in manjši delež vseh ostalih tipov VWB. Razlog za to je lahko majhno število bolnikov, vključenih v raziskavo in ciljana osredotočenost na analizo eksona 28 gena VWF. Bolniki, pri katerih smo opredelili le benigne spremembe, bodo naknadno analizirani z metodo NGS, saj pričakujemo, da se spremembe nahajajo v drugih delih gena VWF. Do sedaj je za bolnike, kjer obstaja sum na VWB, potrditev diagnoze temeljila le na kliničnih podatkih. Z molekularno genetskimi analizami lahko sedaj hitreje in bolj natančno potrdimo diagnozo.

• Language: Slovenian
• Keywords: Von Willebrandova bolezen, Von Willebrandov faktor, genetske spremembe VWF na eksonu 28, parcialni neobdelani psevdogen VWF
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-146456
• Publication date in RUL: 02.06.2023

Secondary language

• Language: English
• Title: Identification of genetic changes in patients with type 2 von Willebrand disease

Abstract

Von Willebrand disease is associated with disease causing variants in the VWF gene and is the most common inherited blood clotting disorder. Molecular genetic analysis of the VWF gene is extremely complex and has not yet been introduced in the general laboratory practice in Slovenia. The reason for this is the fact that both VWF gene and its pseudogene (VWFP1) are highly polymorphic, which interferes with the molecular genetic analysis, including Sanger sequencing and next-generation sequencing (NGS). The purpose of the master's thesis is to evaluate the genetic causes of VWB type 2 in the Slovenian patients. In the study we included 26 patients suspected of having VWB type 2. We introduced an approach enabling specific amplification of exon 28 of the gene VWF and not pseudogene VWFP1. Amplified fragment was then sequenced using Sanger approach. According to the ACMG classification, detected variants were classified as pathogenic, probably pathogenic, variants of unknown significance (VUS) and benign. All but benign changes were further investigated and their clinical significance was determined. Finally, we compared the detected variants and phenotypes of patients with the phenotypes described in other populations. The frequency of VWB type 2A in our group of patients with VWB type 2 was 25% and with type 2B 12.5%. We did not detect any variants in the patients that would be associated with VWB type 2M and 2N. Compared to other populations, the frequency of VWB type 2A in Slovenia is higher and the frequency of other types is lower. The reason might be low number of patients included in the study and experimental design of the study focusing on exon 28 of the VWF gene. Patients where only benign variants were detected will be subsequently analyzed using the NGS approach, as we expect disease causing variants to be located in other parts of the VWF gene. Until now, for patients with suspected VWB, confirmation of the diagnosis was based only on clinical data. With molecular genetic analyses, we can now confirm the diagnosis more quickly and more accurately.

• Keywords: Von Willebrand disease, Von Willebrand factor, genetic variants of VWF gene on eksone 28, partial unprocessed pseudogene VWF