DNA sequencing

Genomic DNA from 34 patients underwent WES. Purified DNA was fragmented, end-repaired, A-tailed, and underwent adaptors ligation and DNA fragments enrichment. Next-generation sequencing was carried out on HiSeq 4000 System (Illumina). Sequencing analysis was performed in all patients using an in-house developed analytical pipeline [18]. The sequencing reads were mapped to the GRCh37/hg19 human reference sequence using the Burrows-Wheeler Aligner (BWA)-MEM alignment algorithm. The BAM files were manipulated by Picard. HaplotypeCaller was used to call potential variant sites. The annotation and filtration of gene variants, including de novo variants, compound heterozygotes, and recessive inherited variants, were generated based on Gemini (version 0.19.1). The functional assessments, including functional prediction algorithms, conservation scores, and ensemble scores, were computed using GERP +  + [19], CADD [20], SIFT [21], and Polyphen-2 [22]. PCR-Sanger sequencing was performed to validate the candidate disease-related variants detected by WES based on Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The PCR program was followed: 95 °C for 3 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s (38 cycles); 72 °C for 8 min. The sequencing results were aligned to reference sequences through CodonCode Aligner (version 6.0.2.6; CodonCode, Centerville, MA, USA).