Study subjects
In this study, we recruited a total of 309 patients with complex CHD. These patients were diagnosed by echocardiography or cardiac catheterization or underwent cardiac surgery at the Shanghai Xinhua Hospital. The patients included 191 males and 118 females (Table 1). Patients with known syndromic CHDs or chromosomal abnormalities were excluded from our study. The controls were 200 population-matched healthy children controls without heart disease. The study protocol was reviewed and approved by the Xinhua hospital Ethics Committee. Both parents and legal guardians of the patients and healthy controls provided signed informed consents. Subsequently, peripheral blood was collected for DNA extraction. The genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) and was stored at -80°C.
Target sequencing and analysis
Genomic DNA was sequenced by target-sequencing technology using the Illumina HiSeq 2000 platform for variants in KLF13 (GenBank accession number NC_000015.10, NM_015995.3) and several other cardiac transcriptional factors involved in cardiovascular development (GATA4, TBX5, TBX1, GATA6, GATA5 and so on). Then, Sanger sequencing was performed to validate all the candidate variants. To evaluated the protein characteristics of nonsynonymous variants, we used SIFT (http://sift.jcvi.org/), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), and Mutation Taster (www.mutationtaster.org/). Amino-acid substitutions were used to predict the damaged variants when the score was ≤ 0.05 in SIFT or ≥ 0.85 in Polyphen-2. KLF13 protein sequences from Homo sapiens (human), Mus musculus (house mouse), Bos taurus (cattle), Capra hircus (goat), Pan troglodytes (chimpanzee), Xenopus laevis (frog) and Sus scrofa (swine) were downloaded from the Universal Protein (UniProt) database (http://uniprot.org/) and were aligned with Clustal X software.
Plasmid construction and site-directed mutagenesis
The KLF13 and TBX5 cDNA plasmids were purchased from Genomeditech. Site-directed mutagenesis for the KLF13 point mutations, c.467G>A (S156N) and c.487C>T (P163S), were constructed according to the protocol provided by the Site-Directed Mutagenesis Kit (Stratagene, USA). Then, the mutated sites were confirmed with Sanger sequencing. The luciferase human B-type natriuretic peptide (BNP) promoter was constructed as previously described [19].
Cell cultures and transfection
For cell culture experiments, 293T cells were used for protein extraction and immunofluorescent staining. NIH 3T3 cells were used for Luciferase assays and were maintained in growth medium (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Plasmids were transfected with FuGene HD (Promega, USA) according to the manufacturer’s protocol, 24 hours after cells were seeded.
Luciferase assays
NIH3T3 cells were seeded onto a 24-well plate, and 600 ng of BNP luciferase reporter vector, different dosages (25, 50, 100, 200, and 300 ng) of wild-type/variant KLF13 plasmids, and pCMV-Tag2B vector with or without TBX5 plasmids were transferred with FuGene HD. The luciferase activity was measured by the Dual-Gloluciferase assay system (Promega), following the manufacturers protocol after 48 hours of transfection. Firefly luciferase activities were reported as fold activation. All the experiments were repeated at least three times in duplicate.
Immunofluorescence
For myc staining, 293T cells were harvested 48 hours after transfection with wild type-KLF13 and variants. After fixation with 4% paraformaldehyde at room temperature for 10 minutes, permeation with 0.3% TritonX-100 for 15 minutes, and blocking with 5% BSA for 1 hour, cells were incubated with the primary antibody (1:500, Rabbit Polyclonal, anti-myc, 16286-1-AP, proteintech, USA) overnight at a temperature of 4°, followed by incubation with the conjugated secondary antibody, anti-rabbit Alexa Fluor® 488 (1:500, R37116, Life technologies, USA) at room temperature in the dark for 1 hour. DAPI (Vector Laboratories, USA) was used for nuclear staining. The images were acquired using fluorescence microscopy at 200X magnification with Image Pro Plus software.
Western Blotting analysis
The cells 293T were harvested at 48 hours after transfection with wild type-KLF13 or variants. The protein was extracted as previously described [15]. A total of 50 µg of the extract was subjected to 10% SDS-PAGE and was transferred onto nitrocellulose membranes and blocked with skim milk (5%) in TBST (0.1%) at room temperature for 2 hours, using gentle agitation during the process. Then, the membranes were incubated with the primary antibody (myc: 1:1000, Rabbit Polyclonal, anti-myc, 16286-1-AP, proteintech, USA; GAPDH: 1:1000, Rabbit monoclonal, anti-GAPDH, ab181602, Abcam, USA) overnight at a temperature of 4°. Following the incubation with the primary antibody, horseradish-peroxidase-conjugated secondary antibody (1:1000, 111-005-003, Jackson Immuno Research, USA), and Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) were used to visualize the chemiluminescent immunodetection.
Coimmunoprecipitation
The cells 293T were transfected with wild type-KLF13 or variants and TBX5 plasmids using FuGene HD according to the manufacturer’s guidelines. Protein extracts were incubated overnight with anti-myc antibodies (1:100, Rabbit Polyclonal, anti-myc, 16286-1-AP, proteintech, USA) coupled with magnetic beads. Bound proteins were revealed with anti-TBX5 (1:1000, Rabbit Polyclonal, anti-TBX5, 42-6500, Invitrogen, USA) antibodies by Western blotting.
Statistical analysis
Data are reported as means ± SEMs. Two-way analysis of variance (ANOVA) was used to compare differences among groups by SPSS. A P value < 0.05 was considered to indicate statistical significance.