Pedigree and clinical diagnosis
Two Chinese patients with oligodontia, aged 10 and 11, were identified in the Department of Endodontics of Nanfang Hospital (Guangdong, China). Panoramic radiographs confirmed the diagnosis of non-syndromic hypodontia. A pedigree construction was created by conducting clinical examinations of the available family members and by conducting interviews. A total of 6 family members participated in this study. Tooth agenesis could be traced back three generations. All subjects gave informed consent and the study was approved by the Ethics Committee of Southern Medical University (No. NFEC-2021-137).
Identification of mutations
To identify disease-associated mutations, we used a standard phenol/chloroform extraction method to extract genomic DNA from the peripheral blood of the proband individual and his family members. Screening of pathogenic mutations was performed using polymerase chain reaction (PCR) amplification and by sequencing the complete exons and exon–intron boundaries of EDA. PCR products were submitted to Invitrogen (Shanghai, China) for sequencing. Then, to confirm the causative mutation, co-segregation analyses were performed for all family members.
Construction of EDA expression vectors, site-directed mutagenesis and vector 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 lentiviruses and the control lentivirus (Ctrl), which were synthesized by GeneChem (Shanghai, China), to establish a stably transfected cell line in the subsequent studies.
Cell culture and odontoblastic differentiation
Isolation of hDPSCs was performed as described elsewhere [26]. For the odontoblastic differentiation experiments, the cells were cultured in odontogenic medium (OM), consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Invitrogen, NY, USA), 10% foetal bovine serum (FBS, Gibco), 50 mg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 5 mM b-glycerophosphate (Sigma-Aldrich), and 10 nM dexamethasone (Sigma-Aldrich).
Cell infection
We overexpressed WT and MUT Flag-EDA samples in hDPSCs with lentivirus following the manufacturer’s instructions. hDPSCs were seeded in 12-well plates at a density of 105 cells/well and grown for 24 h. The cells were infected with a multiplicity of infection (MOI) of 20 in the presence of 5 mg/mL polybrene for 10 h at 37°C and 5% CO2.
Western blot analysis
Cells were harvested and lysed in RIPA buffer (Beyotime, Nanjing, China) supplemented with protease inhibitors (Beyotime). Total protein (20 μg) was separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham, Little Chalfont, UK). The membranes were blocked for 1 h 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 (1:1000; SAB, China), anti-p-p65 (1:1000; SAB, China), anti-IkBa (1:1000; SAB, China), and anti-p-IkBa (1:1000; SAB, China) antibodies. The next day, the membranes were incubated for 1 h at 37°C with the corresponding secondary antibodies (Proteintech, China), and the immunoreactive proteins were visualized with an ECL Kit (Beyotime Biotech, Shanghai, China) according to the manufacturer’s instructions.
Quantitative real-time PCR (RT-qPCR)
Quantitative real-time PCR (RT-qPCR) 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 Biotechnology, China) on a LightCycler 480 (Roche, Indianapolis, USA). The DSPP and GAPDH primers have been published elsewhere [26]. The EDA primers were as follows: forward primer, 5′-CTCGAGAAAACCAGCCAGC-3′; and reverse primer, 5′-CAGTCATTGAGCACTCCACC-3′.
Alizarin Red S (ARS) staining
The number of calcium nodules formed by hDPSCs after transfection with EDA lentivirus vector, WT, or MUT EDA was analysed by Alizarin Red S (ARS) staining. After being cultured in OM for 14 days, the cells were fixed using 60% isopropanol (Macklin, China) for 30 min and stained with 1% ARS (Sigma-Aldrich, USA) at room temperature. After washing several times with deionized water, calcium nodules were observed with a microscope (Crystal violet, Amresco, Solon, OH).
Detecting skewing of X inactivation
The XCI pattern was analysed using the human androgen receptor (HUMARA) assay [27], which utilizes the highly polymorphic CAG repeat sequence in the first exon of the androgen receptor (AR) gene. With digestion of the methylation-sensitive restriction enzyme HpaII and differential PCR amplification, the AR gene was used as a marker of skewed XCI. In brief, DNA samples were obtained from the peripheral venous blood of the female carrier (II3) and her affected father (I3). For each sample, 600 ng of DNA was digested with HpaII. Digested products, together with non-digested DNA, were used as templates for the amplification of the AR polymorphic repeat sequence using the following fluorescence primers: AR forward, 5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′; and AR reverse, 5′-TCCAGAATCTGTTCCAGAGCGTGC-3′. We identified the digestion efficiencies with MIC2 as an internal reference. The amplification products of MIC2 could be observed when the DNA was not completely digested. The primers were as follows: MIC2F forward, 5′-AGAGGTGCGTCCGATTTTTCCC-3′; and MIC2F reverse, 5′-ACCGCCGCAGATGGACAATT-3′. The products were analysed by capillary electrophoresis (Beckman Coulter, USA).
Bioinformatics
To further confirm the mutant EDA function, 3D structures of the 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
Statistical analyses were performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). The statistical significance between the two groups was determined using an independent samples t test, and significance between more than two groups was analysed by one-way- ANOVA. P < 0.05 was considered significant.