Qiamp blood mini kit

Genomic DNA of the patient and her parents was extracted from peripheral blood using the QIAmp Blood minikit (Qiagen, Venlo, the Netherlands). Whole-exome sequencing (WES) was applied to detect mutation. All samples were sequenced on an Illumina platform by Euler Genomics (Beijing, China). Synonymous changes and single nucleotide polymorphisms with minor allele frequency (MAF) higher than 5% were removed. Clinical significance and pathogenicity of the identified variant was interpreted according to the guidelines set by the American College of Medical Genetics (ACMG) [25 (link)], and was further validated by Sanger sequencing. Three-dimensional model of the variant compared with the wild-type was predicted by Swiss model. The human SATB2 cDNA and the mutant SATB2 cDNA with Flag-tag were cloned into pcDNA3.1 expression vector respectively.

To determine copy numbers of pfmdr1, plasmepsin II, and III, qPCR was performed on genomic DNA (extracted with QIAmp Blood Mini Kit, Qiagen, Hilden, Germany) as previously described^{47 (link)} with the following modifications: amplification reactions were done in MicroAmp 384-well plates in 10 μl SYBR Green master mix (Applied Biosystems, Foster City, CA), 150 nM of each forward and reverse primer and 0.4 ng template. Forty cycles were performed in the Applied Biosystems ViiA^TM 7 Real-time PCR system (Life Technologies, Carlsbad, CA). pfmdr1 forward (5’-TGCATCTATAAAACGATCAGACAAA-3’) and reverse primers (5’-TCGTGTGTTCCATGTGACTGT-3’) were designed after Price, et al.^{48 (link)}, whereas β- tubulin forward (5’-CGTGCTGGCCCCTTTG-3’) and reverse (5’-TCCTGCACCTGTTTGACCAA-3’) primers for the endogenous control were designed after Ribacke, et al.^{47 (link)}. Two primer sets were designed for plasmepsin II: 1st set: forward (5’-TCCTTGGTTTAGGATGGAAAGA-3') and reverse 5’-CCACCAATGGTTAAGAATCCTG-3’) 2nd set: forward (5’-CCATTGGTGGTATTGAAGAAAGA-3’) and reverse (5’-TTTCCAACGTGTGCATCTAAA-3’); and, one set for plasmepsin III: forward (5’-GGTAGTGAGTTTGATAATGTGG-3’) and reverse (5’-CACAAGACTCTGATGTACA-3’). Technical replicates were run in quadruplicates. Copy numbers were considered increased ( > 1) when the average of three biological replicates was above 1.6.

To extract DNA, blood clots were broken and homogenized in a conical tube using the pipette tip and mixed with 1x lysis buffer (Qiagen QIAmp Blood mini kit) and incubated at 4 °C for 30 min. After centrifugation at 3000 rpm for 20 min, the supernatant was decanted and 1x lysis buffer was added again and centrifuged at 3000 rpm for 20 min. After the addition of 500 µL of saline EDTA (ethylenediaminetetraacetic acid), 400 µL of 0.2 M sodium acetate, 300 µL of 5% SDS (sodium dodecyl sulfate) and proteinase K, the pellets were incubated overnight at 37 °C. The DNA in this lysate was purified using phenol:chloroform:isoamyl alcohol (25:24:1) mixture, followed by precipitation with ethanol and re-suspension in 20 µL of TE (Tris EDTA) buffer. The DNA was stored in −20 °C till further molecular work up.

Killer immunoglobulin-like receptors genotyping was performed on DNA extracted from 400 μl of blood using QIAmp Blood Mini kit (Qiagen) with the KIR Genotyping SSP kit (Invitrogen) according to manufacturer’s instructions. The AA haplotype was defined as KIR2DL5A^neg/KIR2DL5B^neg/KIR2DS1^neg/KIR2DS2^neg/KIR2DS3^neg/KIR2DS5^neg/KIR2DS1^neg.

Genomic DNA was extracted from 200 µL blood samples with a QIAmp Blood Mini Kit (QIAGEN) according to the manufacturer’s instructions. The P. malariae aldolase gene was amplified using primers ALf (5′-CAGGCATCAAGCGCAGACTA-3′) and ALr (5′-TAAAGCCCATGGGTGAGGTC-3′) [13 (link)], while the P. malariae LDH gene was amplified using primers LDHf (5'-ACTTTACAGCCGCCCATTCC-3') and LDHr (5′-CCTTCATTCTCTTCGTTTCAGCA-3′) [14 (link)]. Conventional PCRs were conducted in 20 µl volumes with a KOD-401 kit. The products were sequenced by Shanghai exsyn-bio Technology Co., Ltd., and the sequence results were analysed with MEGAX.