Ethical approval and subjects
This study was approved by the Clinical Research Ethics Committee of Peking University First Hospital. The patient was recruited from our clinic and written informed consent was obtained from her guardians in adherence to the Declaration of Helsinki Principles.
Mutational analysis and Quantitative real-time PCR
The peripheral blood sample and biopsy samples of affected skin tissues from the patient were collected. Skin lesions were separated into epidermis and dermis according to a standard method. We performed whole-exome sequencing (WES) using genomic DNA extracted from the dermis with vascular malformation lesions. The criteria for selection of candidate variants was as follows: (a) total read depth across the position ≥ 20, mutant reads ≥ 5, mutant reads frequency ≥ 1%. (b) nonsynonymous variants; (c) variants absent or with a minor allele frequency < 1% in any of the public databases (the [1000G], [ExAC], [ESP 2500 and 6500] and the [gnomAD]); (d) variants predicted to be “damaging”, “probably damaging” or “disease-causing” by at least one in silico tools, including SIFT, Mutation Taster, PolyPhen and GERP. Sanger sequencing was used to confirm the candidate pathogenic mutation found by exome sequencing.
Total RNA from the individual’s dermis of the affected skin samples was extracted using TRIzol reagent (Invitrogen) and equal amounts of RNA from each sample were reverse transcribed to cDNA according to the manufacturer’s instructions. To access the consequence of the mutation, we performed quantitative real-time PCR analysis of PIK3CA using the cDNA samples. Amplicons were quantified with Power SYBR Green PCR Master Mix (ABI) using a CFX Connect Real-Time System (Bio-Rad), and expression levels were normalized to those of GAPDH.
Cell culture
HEK293 cells were purchased from ATCC (American Type Culture Collection, USA) and incubated in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum containing 100 U/ml of penicillin and 100 U/ml of streptomycin at 37 °C in an atmosphere of 5% CO2.
Plasmids constructions and transfection
For exogenous expression in mammalian cells, human PIK3CA cDNA was cloned into pLenti-CMV vector encoding a C-terminal 3 × Flag tag. The point mutations in PIK3CA (c.501G>C, c.1624G>A, c.3140A>G, and c.3206_3207insG [Arg115Pro, Glu542Lys, His1047Arg, and X1069Trpfs*4]) were generated following the manufacturer’s protocol of C214 Mut Express® II Fast Mutagenesis Kit V2. HEK293 cells were seeded into six-well plates at a density of 4 × 105 cells per ml. Transfections with 3 μg of plasmids of empty vector, PIK3CA-wildtype (PIK3CA-WT), PIK3CA-Arg115Pro, PIK3CA- Glu542Lys, PIK3CA- His1047Arg, and PIK3CA-X1069Trpfs*4 were performed in the six-well plates when cell confluence reached 80%.
Western blot analysis
Forty-eight hours after transfection, cells were washed twice in PBS and lysed in lysis buffer. Total proteins were extracted from the cultured cells and the protein concentration was measured using the Bradford method according to the manufacturer’s recommendations. All the protein samples were heated with loading sample buffer and reducing agent buffer( NuPAGE ) for 10min at 70°C. The nitrocellulose filter (NC) membranes transferred with the proteins were blocked with 5% skim milk in TBST for 1h at room temperature and then incubated with first antibodies overnight at 4°C. Specific first antibodies: FLAG, phospho-AKT Ser473, phospho-AKT Thr308, AKT, ERK, phospho-ERKThr202/204, STAT1, phosphor-STAT1Tyr701 were bought from Cell Signaling Technology. Second antibodies: horseradish peroxidase (HRP)-linked anti-mouse/rabbit antibody. GAPDH was used as a control.
Inhibitors treatment
We also tested the inhibitory effects of three inhibitors (BYL719, ARQ092, rapamycin) related to the PI3K/AKT signaling pathway. HEK293 cells were seeded into six-well plates and then transfected following the standard method. Then cells were then treated with full media containing different concentrations of three inhibitors respectively: 10μM BYL719, 5μM ARQ092, 15μM rapamycin 48h after transfection. Protein samples were collected from cell lysates 1 hour after treatment with inhibitors and Western blot analysis was also performed to assess inhibitory effects.