Balkan Journal of Medical Genetics

SINGLE-NUCLEOTIDE POLYMORPHISMS IN EXONIC AND PROMOTER REGIONS OF TRANSCRIPTION FACTORS OF SECOND HEART FIELD ASSOCIATED WITH SPORADIC CONGENITAL CARDIAC ANOMALIES
Wang E, Fan X, Nie Y, Zheng Z, Hu S,
*Corresponding Author: Shengshou Hu M. D., Cardiac Surgery Department, Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Xicheng District, Beijing, 100037, China, Tel & Fax: 86-010-88322325 E-mail: shengshouh@sina.cn
page: 39

MATERIALS AND METHODS

Patient information From January 13, 2012 to May 5, 2012, a total of 383 patients were enrolled in this study. These patients were suffering from sporadic CHD and were scheduled for surgery in our hospital (Table 1). The average age of the patients was 1 year old (3 months - 9 years). Patients were classified into simple CHD [ventricular septal defects (VSD), atrial septal defects (ASD), and patent ductus arteriosus (PDA)] (33%), right ventricular outflow tract obstruction (RVOTO) [tetralogy of Fallot (TOF), pulmonary atresia (PA), and pulmonary stenosis (PS)] (58%), and single ventricle (SV) (9%) (Table 1). A total of 383 healthy children were also recruited from our hospital as a control group. No significant differences were observed in age or sex between the CHD patients and control subjects (Table S1, S2). The diagnosis and inclusion and exclusion criteria for the subjects are described in the “Methods section” of the supplementary material. The study complied with the 1964 Declaration of Helsinki and its subsequent amendments and was approved by the Medical Ethics Committee of Fuwai Hospital. All patients or their legal guardians signed informed consent forms. DNA extraction and genotyping In all subjects, the extraction of genomic DNA from leukocytes was performed with a Wizard® Genomic DNA Purification Kit (Promega, WI, USA). Ten SNPs in exonic and promoter regions from 4 genes (GATA5, SMYD1, TBX20, and MEF2C) (from unpublished sequencing data for CHD) were genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI- TOF-MS) in both CHD patients and control subjects (Figure 1, 2). The steps of MALDI-TOF-MS included multiplex polymerase chain reaction (PCR), amplification, shrimp alkaline phosphatase digestion, IPLEX primer extension, resin cleaning, MALDI-TOF-MS analysis, and data analysis (14). Analyses were repeated in 10% of randomly selected samples for quality control. Plasmids, site-directed mutagenesis, cell transfection, and luciferase assays MEF2C: rs80043958 A>G, MEF2C: rs304154 A>G, and TBX20: rs336284 A>G were all located in promoter elements. The promoter fragments of MEF2C containing the A allele of rs80043958 or rs304154 and the TBX20 promoter fragment containing the A allele of rs336284 were amplified from genomic DNA. The PCR products were subcloned into the KpnI/XhoI restriction sites of the GV238-basic vector (GeneChem, Shanghai, China). Transcription factors included ZFX (GenBank accession no. NM_001330327), CEBPA (GenBank accession no. NM_001287424), HLTF (GenBank accession no. NM_003071), FOXC1 (GenBank accession no. NM_001453), and GATA1 (GenBank accession no. NM_002049) were also amplified and subcloned into the GV141-basic vector. Plasmids carrying the corresponding G allele were generated by site-directed mutagenesis with the MutanBEST kit (Takara, Berkeley, CA, USA) to ensure a uniform backbone sequence. All recombinant clones were verified by DNA sequencing. The human embryonic kidney cell line HEK 293T (4×105) was seeded in 24 well culture plates. After 24 h, HEK 293T cells were transfected with 1.0 μg of the wild-type promoter or mutant promoter and the corresponding transcription factors, according to the manufacturer’s instructions. After an additional 24 h of culture, the transfected cells were assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). There were eight experimental groups for rs80043958 and rs304154. For rs336284, there were four experimental groups. Each luciferase assay was performed in triplicate. Statistical analyses The means ± standard deviations (SD) were used for the continuous variables. Continuous variables were compared between the two groups by Student’s t test. Pearson’s χ2 test or Fisher’s exact test was used to compare the categorical variables between the two groups. The odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were estimated for the risk of CHD. Differences were considered significant if p<0.05. The statistical analyses were performed using the SPSS version 17.0 software package (SPSS, Inc., Chicago, IL, USA).

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