Whole-exome sequencing (WES)

WES was used to detect the sequence variants in the samples of the probands (16). Briefly, the sequences of the target region were enriched using the Agilent Sure Select Human Exon Sequence Capture kit (Agilent Technologies, Inc.). The DNA libraries were tested using quantitative PCR, where the size, distribution and concentration were determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The DNA of ~150 bp paired-end reads was sequenced using the NovaSeq6000 platform (Illumina, Inc.), taking ~300 pM of DNA per sample using the NovaSeq Reagent kit. Sequencing raw reads (quality level of Q30%>90% and the criteria for quality listed at https://www.illumina.com/science/technology/next-generation-sequencing/plan-experiments/quality-scores.html) were aligned to the human reference genome (accession No. hg19/GRCh37) using the Burrows-Wheeler Aligner tool (bwa-0.7.17.tar.bz2) (17), following which, duplicate PCR products were removed using the program Picard v1.57 (https://github.com/broadinstitute/picard). Variant calling was performed using the Verita Trekker^® Variants Detection system (v2.0; Berry Genomics Co., Ltd.) and Genome Analysis Tool kit (https://software.broadinstitute.org/gatk/). Then, variants were annotated and interpreted using the ANNOVAR (v2.0) (18) and Enliven^® Variants Annotation Interpretation systems (Berry Genomics Co., Ltd.), according to the guidelines by ACMG (American College of Medical Genetics and Genomics) (19). To assist in the interpretation of variant pathogenicity, the present study referred to three frequency databases, namely ExAC_EAS (http://exac.broadinstitute.org), gnomAD_exome_EAS (http://gnomad.broadinstitute.org), 1000G_2015aug_eas (https://www.internationalgenome.org) and Human Gene Mutation Database Pro v2019 (https://www.hgmd.cf.ac.uk/ac/index.php). The Revel score (a combined method of pathogenicity prediction) (20) and pLI score (representing the tolerance for truncating variants) were also employed.