Contribution of complete sequence analysis of von Willebrand factor and coagulation factor VIII genes in the diagnosis of von Willebrand disease and distinction from mild haemophilia A

Autor: Lapić, Ivana
Přispěvatelé: Zadro, Renata, Rogić, Dunja
Jazyk: chorvatština
Rok vydání: 2022
Předmět:
Popis: Višestruka uloga von Willebrandova faktora (VWF) u hemostazi, složenost njegove strukture i veliki broj mutacija u različitim dijelovima gena za VWF čine von Willebrandovu bolest (VWB) klinički izrazito heterogenim poremećajem, a njegovu pravilnu dijagnostiku i razlikovanje od blage hemofilije A otežanima. Cilj ovog istraživanja bio je primijeniti cjeloviti pristup dijagnostici VWB-a u Republici Hrvatskoj koji uključuje globalne i visokodiferentne koagulacijske pretrage kojima su ispitana funkcionalna i strukturna svojstva VWF-a te molekularnu analizu svih sljednih varijanti u genima za VWF i faktor zgrušavanja VIII (FVIII) sekvenciranjem sljedeće generacije (NGS) i dodatnim pretraživanjem velikih delecija i duplikacija metodom višestrukog umnažanja vezanih sondi. Istraživanje je obuhvatilo 83 ispitanika, a mutacije u genu za VWF nađene su kod njih 48. Na temelju rezultata koagulacijskih pretraga i genetičke analize, 19 ispitanika klasificirano je kao tip 1 VWB-a, šest kao tip 3, dok je među 23 ispitanika s tipom 2 VWB-a 15 imalo tip 2A, sedam tip 2B, a tip 2N potvrđen je u jednom slučaju. Heterozigotni genotip utvrđen je kod 38 ispitanika, sedam su bili složeni heterozigoti i tri homozigoti. Ukupno je utvrđeno 36 različitih mutacija u genu za VWF, od kojih su 13 novootkrivene. Mutacije u genu za FVIII nađene su kod petero ispitanika, pri čemu je u dva muška homozigota postavljena dijagnoza blage hemofilije A, dok su tri ženske ispitanice heterozigoti i smatraju se nositeljicama hemofilije A. Kod jedne je ispitanice uz mutaciju u genu za FVIII nađena i mutacija u genu za VWF. Dvije su mutacije u genu za FVIII novootkrivene. Kod 30 (36 %) ispitanika nisu nađene mutacije u genima za VWF i FVIII, pri čemu je devet ispitanika zbog aktivnosti VWF-a ispod 50 % svrstano u kategoriju tzv. ꓹꓹniskog VWF-a“. Ovo istraživanje upozorilo je na znatnu heterogenost genetičke osnove VWB-a u hrvatskoj populaciji, a istodobno sekvenciranje gena za VWF i FVIII tehnologijom NGS omogućilo je jednoznačno postavljanje dijagnoze VWB-a te diferencijalno dijagnostičko razlučivanje od blage hemofilije A. Time je osigurana osnova za pravilno liječenje i skrb za bolesnike te su razjašnjeni molekulsko-patofizološki mehanizmi koji su u podlozi poremećaja krvarenja kod obrađenih bolesnika. Multiple functions of von Willebrand factor (VWF), its structural complexity and numerous mutations throughout the whole VWF gene impact the clinical heterogeneity of von Willebrand disease (VWD) and make its accurate diagnosis and distinction from mild haemophilia A challenging. The aim of the present study was to introduce a comprehensive laboratory diagnostic approach that included screening and specific coagulation assays, as well as molecular analysis of VWF and coagulation factor VIII (FVIII) genes by means of next-generation sequencing (NGS) and additional targeted screening for deletions and duplications using the multiplex ligation-dependent probe amplification method. Of the 83 study participants, 48 were identified with disease-associated mutations in the VWF gene, of whom 19 were classified as type 1 VWD, six as type 3 VWD, while among the 23 patients with type 2 VWD, 15 were assigned as type 2A, seven as type 2B, and in one case type 2N VWD was confirmed. Heterozygous genotype was present in 38 patients, compound heterozygosity in seven patients while three patients were homozygous for mutations within the VWF gene. In total, 36 distinct disease-associated mutations were found within the VWF gene, of which 13 were novel. Four study participants were identified with mutations within the FVIII gene only, while one female participant had mutations both in VWF and FVIII genes. The two siblings who were homozygous for a mutation in the FVIII gene were diagnosed with mild haemophilia A, while the three heterozygous females were classified as carriers of mild haemophilia A. Two mutations within the FVIII gene were novel. Of the remaining 30 (36 %) study participants without mutations within the VWF and FVIII genes, nine had VWF activity below 50 % and were classified as “low VWF“. The present study revealed considerable genetic heterogeneity among patients with VWD in Croatia. The application of simultaneous sequencing of whole VWF and FVIII genes by means of NGS was proven as a valid approach for differential diagnosis of VWD subtypes, as well as for unambiguous distinction of VWD from mild haemophilia A. This study provided basis for appropriate patient treatment and care, as well as enhanced the understanding of the underlying molecular pathophysiology of the bleeding phenotype in the evaluated patients.
Databáze: OpenAIRE
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