Application of massive parallel sequencing searching for rare genetic variants associated with thrombophilia - implementation to clinical practice.
P. Vrtel, L. Slavik, R. Vodicka, R. Vrtel, J. Ulehlova, J. Prochazkova, A. Hlusi, M. Palova, M. Prochazka (Olomouc, Czech Republic)
Time: 17:15 - 18:15
Objective: Thromboembolism have multifactorial etiology but one of the main causes is thrombophilia. Really common and severe cause of thrombophilia is deficiency of protein C, S or antithrombin III (PC, PS, AT III). The genetic cause are mutations without mutational hot-spots areas in the studied genes. Therefore it is appropriate to use methodology of massive parallel sequencing (MPS) and expanded molecular genetic analysis for detection of genetic cause of deficiency of those proteins. Selected proteins are encoded by genes: PROS, PROC, SERPINC1. In these genes have been described more than 800 mutations whose clinical manifestation may be a lack or loss of function of the gene product, which are involved in the inhibition of coagulation factors. Pathogenic mutations in these genes exhibit similar risk of thrombosis as routinely tested mutations of FV Leiden and FII Prothrombin (G20210A).
Methods: MPS was carried out by Ion Torrent PGM platform. We used Ampliseq Designer for design of multiplex with 100% coverage of coding sequences and exon / intron border areas. The data were processed by Torrent Suite programs - Ion Reporter and Next Gene - available database of clinical variants (ClinVar, HGMD).
Results: In the first run there were examined 10 patients. They were selected for repeatedly detected reduced levels of protein C or S, at the same time they were excluded for secondary etiology of that condition. We have revealed six missense mutations in PROS1(2) and PROC (4) so far.
Conclusion: MPS offers complex tool for identification genetic causes of severe thrombophilia states applicable to laboratory and clinical praxis. Further it will be useful to add MLPA analysis for patients where MPS doesn´t reveal any genetic cause. MLPA should improve testing for better detection of large rearrangements in examined genes. Supported by LF-001-2017 and MH CZ – DRO (FNOl, 00098892)