Pedigree and clinical diagnosis
Two Chinese oligodontia patients were identified in the Department of Endodontics at the School of Stomatology, Southern Medical University. Panoramic radiographs confirmed the diagnosis of non-syndromic hypodontia. A pedigree construction was constructed by clinical examinations of available family members and through interviews. A total of 6 family members participated in this study. Tooth agenesis could be traced back to three generations. All subjects gave informed consent and the study was approved by the Ethics Committee of Southern Medical University.
Identification of mutations
To identify disease-associated mutations, we extracted genomic DNA from the peripheral blood of the proband and her family members by a standard phenol/chloroform extraction method. Screening of pathogenic mutations was performed using polymerase chain reaction (PCR) amplification and sequencing the complete exons and exon–intron boundaries of EDA. Details of primers and PCR conditions have been published elsewhere.
Construction of EDA expression vectors, site-directed mutagenesis and their lentivirus package
Full-length human EDA was cloned into the expression vector pcDNA3.1 (Invitrogen), and EDA-Wild Type (WT) was obtained. The c.1013C>T mutant (MUT) was generated using the following primers: forward, 5’-ACACGCAGCATCGAGATGGGCAAGACCAACTAC-3’; and reverse, 5’-GTAGTTGGTCTTGCCCATCTCGATGCTGCGTGT-3’, which replaced threonine (Thr) with methionine (Met). Then the plasmid vectors were packaged into lentivirus, which was synthesized by Genechem (Shanghai, China), including control lentivirus (Ctrl), in order to establish a stable transfected cell line in the following studies.
Cell culture and their odontoblastic differentiation
Isolation of human dental pulp stem cells (hDPSCs) was performed as described elsewhere [23]. For odontoblastic differentiation experiments, the cells were cultured in an odontogenic medium (OM), consisting of dulbecco’s modified Eagle’s medium (DMEM), 10% of fetal bovine serum (FBS), 50mg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 5mM b-glycerophosphate (Sigma-Aldrich), and 10nM dexamethasone (Sigma-Aldrich). The procedure to acquire human tissues was approved by the Ethics Committee of Southern Medical University and Nangfang Hospital of Stomatology.
Cell infection
We overexpressed WT or MUT Flag-EDA in DPSCs with lentivirus following the manufacturer’s instructions. The DPSCs were seeded in 12-well plates at a density of 105 cells/well and grown for 24h. The cells were infected at a multiplicity of infection (MOI) of 20 in the presence of 5mg/ mL polybrene for 10h at 37°C and 5% CO2.
Western blot analysis
Cells were harvested and then lysed in RIPA buffer (Beyotime, Nanjing, China) supplemented with protease inhibitors. Total protein (20 μg) was separated by 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham, Little Chalfont, UK). The membranes were blocked for 1h with 5% skim milk and incubated overnight at 4°C with anti-FLAG,anti-GAPDH (1:1000; proteintech, China), anti-DSPP (1:1000; Santa Cruz Biotechnology), anti-p65,anti-p-p65,anti-IkBa,anti-p-IkBa (1:1000; SAB,China) antibodies. The next day, the membranes were incubated for 1h at 37°C with the corresponding secondary antibodies (Proteintech, China), and the immunoreactive proteins were visualized with the ECL Kit (Beyotime Biotech, Shanghai, China) according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
Quantitative RT-PCR was applied to examine the expression of EDA and dentin sialophosphoprotein (DSPP). Total RNA was reverse-transcribed using the Prime Script First Strand cDNA Synthesis Kit (TaKaRa Biotechnology, China), and RT-qPCR was carried out with SYBR Premix DimerEraser (TaKaRa) on a LightCycler 480 (Roche, Indianapolis, USA). The DSPP primers have been published elsewhere.
Alizarin Red S staining (ARS)
The number of calcium nodules formed by hDPSCs after transfection of EDA lentivirus vector, WT, or MUT EDA was analyzed by Alizarin Red S staining. After culture in the osteo/odontogenic medium (complete culture medium containing 50mg/mL ascorbic acid, 5mM b-glycerophosphate, and 10nM dexamethasone (Sigma-Aldrich)) for 14 days, the cells were fixed using 60% isopropanol and stained with 1% ARS (Sigma, USA) at room temperature.
Detecting Skewing of X Inactivation
The X-chromosome inactivation pattern was analyzed using the human androgen receptor (HUMARA) assay [24], which utilize the highly polymorphic CAG repeat in the first exon of the androgen receptor gene (AR). The AR gene has been used as a marker of skewed X chromosome inactivation through differential PCR amplification following digestion with the methylation-sensitive restriction enzyme HpaII. In brief, DNA samples were obtained from the peripheral venous blood of the female carrier (II3) and her affected father (I3). For each sample, 600ng of DNA was digested with HpaII. Digested products together with non-digested DNA were used as templates for amplification of the AR polymorphic repeat using fluorescence primers: AR, forward: 5’-GCTGTGAAGGTTGCTGTTCCTCAT-3’ and reverse, 5’-TCCAGAATCTGTTCCAGAGCGTGC-3’. And we identified the digestion efficiencies with MIC2 as internal reference. The amplification products of MIC2 can observed when the DNA was not complete digestion. The primers were as follows: MIC2F, forward: 5’-AGAGGTGCGTCCGATTTTTCCC-3’ and reverse, 5’-ACCGCCGCAGATGGACAATT-3’. The products were analyzed by capillary electrophoresis (Beckman Coulter, USA).
Bioinformatics
To further confirm the function of mutant EDA, the 3D structures of WT and mutant EDA were predicted in silico using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/), and the functional effects of the mutant protein were estimated with Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/).
Statistical analysis
Differences between two groups were analyzed using the Student’s t test using IBM SPSS Statistics 20 soft-ware (IBM Corp, NY, USA) and more than two groups were analyzed by one-way-ANOVA. P < 0.05 was considered significant.