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).