2.2 Genetic analysis
Peripheral whole blood samples were collected from patients and their
parents for genetic analyses. Genomic DNA was isolated from peripheral
blood samples of subjects using a whole blood DNA-purification kit
(51104, QIAGEN). WES and bioinformatics analyses were performed on the
patients’ samples. One microgram of genomic DNA was utilized for exon
capture using the Agilent Sure Select Human All Exon V6 Kit (Agilent
Technologies) and sequenced on the Illumina HiSeq X system (Illumina).
Targeted testing of the potentially pathogenic variants in the patients’
parents was performed by Sanger sequencing. PCR amplification was
accomplished with Dyad Polymerase (Bio-Rad Laboratories), and the PCR
products were sequenced on an ABI377A DNA sequencer (Applied
Biosystems). The primers used in PCR analysis were as follows: F,
5’-ACCATTATGAGCTAGCTTTCCTT-3’; and R, 5’-ACAGTAACAACAAAGCCAGGT-3’.