Bioinformatics analysis procedure
We filtered all identified variants through comparison with the
disease-associated variants in the Human Gene Mutation Database (HGMD,
2020.2) and the Online Mendelian Inheritance in Man (www.omim.org),
dbSNP153 (http://www.ncbi.nlm.nih.gov/projects/SNP/), the 1000 Genome
(http://www.1000genomes.org/), the NHLBI Exome Sequencing Project (ESP,
http://evs.gs.washington.edu/EVS/), and GnomAD
(https://gnomad.broadinstitute.org/). To assess the pathogenicity of
missense mutations, we generated the prediction of a variety of
algorithms, including SIFT (https://sift.bii.a-star.edu.sg/), PolyPhen2
(http://genetics.bwh.harvard.edu/pph2/), and Mutation-Taster
(http://www.mutationtaster.org/). The pathogenic and likely pathogenic
genes/variants were defined according to the standards and guidelines of
ACMG (Richards et al., 2015).
Domains of RAPGEF1 were identified by the SMART protein domain
prediction server (http://smart.embl-heidelberg.de/). Multiple sequence
alignments of RAPGEF1 were performed using the ClustalW program
(Thompson, Gibson, & Higgins, 2002). Three-dimensional structural
models of RAPGEF1 were predicted by the I-TASSER web tool
(http://zhang.bioinformatics.ku.edu/I-TASSER/). The visual
representation of models and structural superposition were generated by
the software package of visual molecular dynamics (VMD) (Humphrey,
Dalke, & Schulten, 1996). The mutant stability change (ΔΔG) of variants
of RAPGEF1 were predicted using the STRUM server
(https://zhanglab.ccmb.med.umich.edu/STRUM/). KEGG pathway analysis was
performed on RAPGEF1 and ~700 genes known to be
related to neurodevelopmental disorders (Vissers, Gilissen, & Veltman,
2016) using pathway enrichment analysis tool of Omicshare
(https://www.omicshare.com/tools/home/report/koenrich.html). Three
pathways involving RAPGEF1 were selected. Protein-protein
interaction data between RAPGEF1 and neurodevelopmental disorder-related
proteins were obtained from the STRING database
(https://string-db.org/). The interaction network model was generated
with the package of Cytoscape (Shannon et al., 2003).