WES and mutation screening

To extract genomic DNA, peripheral venous blood was collected from the participants and EDTA was used as the anticoagulant. A blood extraction kit (cat. no. 51304; Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract genomic DNA from leukocytes according to the manufacturer's protocol. Extracted DNA samples were sent to Beijing Nuohe Zhiyuan Technology Co., Ltd. for WES. Genomic DNA was sheared into fragments with a length of 150-200 bp through sonication (17). End-repairing, A-tailing and adaptor ligation, a four-cycle pre-capture PCR amplification and xGen Exome Research Panel (Integrated DNA Technologies, Inc.) enrichment were then performed (17). The sequencing was performed on an Illumina Hiseq Xten platform (Illumina, Inc.) with a mean sequence coverage of ≥90X and a coverage of ≥20X in >95% of the target bases. Cutadapt (https://cutadapt.readthedocs.io/en/stable/) and FastQC (http://darlinglab.org/tutorials/fastqc/) were used to perform quality control. Clean reads were mapped to the human reference genome (University of California Santa Cruz hg19) via Sentieon BWA (version 0.7.15) (18). Duplicate sequence reads were removed using Picard (version 1.85) and variants were detected by GATK (version 3.1) (19). Variants were annotated using ANNOVAR software (version from 14 December 2015; http://annovar.openbioinformatics.org/en/latest/). All exome variants were filtered for allele frequencies <0.001 in the ExAC database (20). The nomenclature of variants was based on the guidelines of the Human Genome Variation Society (21).