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’;