In silico analyses
The deleterious effects of missense variants were assessed with ALAMUT VISUAL (http://www.interactive-biosoftware.com/alamut-visual/), a web-based tool, which allows simultaneous analysis by POLYPHEN-2 (http://genetics.bwh.harvard.edu/pph2) SIFT (http://sift.bii.a-star.edu.sg), MutationTaster (http://www.mutationtaster.org) and GVGD (http://agvgd.iarc.fr/index.php).
The effects of variants at splice junctions were evaluated with ALAMUT VISUAL v.2.8.1 (http://www.interactive-biosoftware.com/alamut-visual/), which allows a simultaneous analysis with the programs SPLICE SITE FINDER-LIKE, MAXENT SCAN, NEURAL NETWORK SPLICE SITE, GENESPLICER, and HUMAN SPLICING FINDER (http://www.cbs.dtu.dk/services/NetGene2/). These tools were used together in accordance with guidelines for using prediction methods (Niroula and Vihinen 2016). Missense variants close to splice sites underwent splice site prediction, too. If three or more of four prediction programs predicted that the mutation under consideration was causing disease, it was accepted as causing hemophilia. For splice site mutations, four out of five prediction programs had to be significant (defined how).