Genomic dna extraction

Genomic DNA extraction (Qiagen, Germany), quality control and quantification measurement (Nanodrop Technologies, USA), ultrasonic fragmentation (Covaris, USA), library construction and target enrichment (SureSelect, Agilent Technologies, USA; Illumina, USA) were conducted according to the manufacturer protocols. High-throughput targeted measurement of gene mutations was performed on an Ion torrent PGM™ (Life Technologies) or MiSeq/HiSeq (Illumina) sequencer platform with an average sequencing depth of 800×. The custom-designed panel consisted of 112 potentially mutated genes which are involved in hematological disorders and are related to the following functional categories: signaling pathways, epigenetic regulators, transcription factors, spliceosomes, cohesin complex, tumor suppressors, NPM1 and others. Single nucleotide variants (SNVs) and short fragment indels in protein coding sequences (CDSs) were analyzed by using Ion Reporter™ and Variant Reporter pipelines and annotated referencing the dbSNP, 1000 Genomes, Polyphen-2 and COSMIC databases. NPM1 (exon 12), FLT3-ITD, and potential complex indels in CEBPA (TAD and bZIP domains) were additionally examined by PCR followed by direct sequencing as previously reported [30 (link)–32 (link)].

The whole blood, which was collected in ethylenediaminetetraacetic acid–containing vacutainer tubes (BD Bioscience; Franklin Lakes, NJ) was subjected to an initial centrifugation procedure to isolate and collect plasma from each patient. Patient-derived PBMCs were then isolated from whole blood using Ficoll-hypaque (Amersham Biosciences; Amersham, UK) density gradient centrifugation as previously described [82] (link). Approximately 5×10⁶ PBMCs were used for genomic DNA extraction (Qiagen; Valencia, CA), which was subsequently used as the substrate for HIV-1 genome amplification.

A total of 760 patients who had been diagnosed with KD were recruited from Guangzhou Women and Children’s Medical Center between January 2012 and December 2017. The diagnosis of KD was made according to the statement of AHA (Newburger et al., 2004 (link)). All patients received a single dose of 2 g/kg IVIG at the diagnosis of KD. IVIG resistance is defined as persistent or recrudescent fever at least 36 h and <7 days after completion of first IVIG infusion (McCrindle et al., 2017 (link)). About 2 ml of whole blood was collected from each participant for genomic DNA extraction (Qiagen, Dusseldorf, Germany). Written informed consent was obtained from the guardians of participants. This study was conducted with the approval form the Guangzhou Women and Children’s Medical Center Ethics Committee (2014073009).