Whole Exome Sequencing of Patients

Peripheral blood samples were collected from the patients and their parents after informed consent was obtained. WES was performed on these patients as mentioned before [12 (link), 13 (link)]. A QIAamp DNA Blood Mini kit® (Qiagen GmbH, Hilden, Germany) was used to isolate genomic DNA. Library was established with an Agilent SureSelect Target Enrichment system (Agilent Technologies, Inc., Santa Clara, CA, USA). And the system of Illumina HiSeq 2000 (Illumina, Inc.) and an Illumina cBot (Illumina Inc., San Diego, CA, USA) were used to sequence and generate clusters. All variants detected were filtered and annotated by Ingenuity Variant Analysis (Ingenuity Systems, Redwood City, CA, USA). Finally, Sanger sequencing was used to confirm the variants detected by WES comparing to the individuals’ parents. The potential pathogenicity of the missense variant was evaluated using three in silico prediction methods: SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and MutationTaster (http://www.mutationtaster.org/ChrPos.html).