2.2 Genetic Analysis
Peripheral blood samples were obtained from all subjects, and the genomic DNA was isolated by DNeasy Blood and Tissue Kit (QIAGEN), according to the manufacturer’s protocol. Next-generation sequencing was carried out using Agilent SureSelectXT Human All Exon Kit and Illumina HiSeq X-TEN. Functional annotation was done using ANNOVAR through a series of databases including 1000Genomes Project, dbSNP, HGMD and ExAC. Next, PolyPhen-2, SIFT, MutationTaster and CADD were used for functional prediction. Targeted testing of the potentially pathogenic variants in the patients’ parents was performed by Sanger sequencing. The primers used in the PCR analysis were as follows: F1, 5’-GACATGAGCTGGCAGAGACAAG-3’; R1, 5’-CAAGGCTCTGGTGGCCTAG-3’; F2, 5’- TGTCATGGCACCAGTCCATG-3’; R2, 5’-GGAGAATTGCTTGAACCTGGCAG-3’; F3, 5’- AATGTGGGAATGTGTAGAGACG-3’; R3, 5’- GCAGATAAGGGAACAAATGGT-3’;