One patient diagnosed with early embryo arrest during common IVF treatment was recruited from the Department of Reproductive Medicine, Xiangya Hospital, Central South University. Participants with normal embryo development in IVF/ICSI cycles were also recruited as the control group. The study was approved by the Institutional Review Board of the Department of Reproductive Medicine, Xiangya Hospital (No.2021005). Informed consent was obtained from all the recruited participants.
Venous blood samples of patients and 100 controls were collected in EDTA blood tubes. Genomic DNA was extracted according to the standard protocol of the QIAamp DNA Blood Midi Kit (QIAGEN). The DNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific).
The Exome was captured using xGen Exome Research Panel v2 (22307, Integrated DNA Technologies, San Diego, California, USA) and sequencing was performed with the Novaseq system (PE150, Illumina, San Diego, California, USA). Sequences were aligned to a human reference sequence (human genome 19, hg19) using Bowtie2. After filtering the unmapped variants, single nucleotide polymorphism (SNP) and insertion and deletion (InDel) polymorphisms were detected using FreeBayes. The variants were annotated using the ANNOVAR software. The frequency of corresponding mutations was determined using the 1000 Genomes Project (1000G), Exome Aggregation Consortium (ExAC) and Exome Aggregation Consortium_East Asian (ExAC_EAS) databases, and the functional effects of the mutations were predicted using the in silico algorithms MutationTaster. The filtering criteria were as followed: (1) variants with an allele frequency lower than 0.1% in the 1000G and ExAC databases; (2) exonic nonsynonymous, splicing, or coding indels; and (3) variants with a previously reported embryogenesis-related function. The American College of Medical Genetics and Genomics (ACMG) was used to assess the mutations. HomozygosityMapper (http://www.homozygositymapper.org/) was used to determine the existence of candidate homozygous variants in the proband .
The targeted variant in PADI6 was amplified by polymerase chain reaction (PCR) using the ProFlex™ Base (Applied Biosystems, Singapore). Specific primers flanking the mutation sites were listed in Additional file 1: Table S1. The mutation variant was then confirmed by Sanger sequencing in the patients and controls.
Evolutionary conservation analysis and molecular modelling
Evolutionary conservation was assessed using Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/). DNA structures was drawn using the Gene Structure Display Server (http://gsds.gao-lab.org/) . The Pfam database (http://pfam.xfam.org/) was used to analyze the protein domains. In addition, wild-type (wt) PADI6 was built using SWISS-MODEL software (https://swissmodel.expasy.org/) in the automated model and mutated PADI6 was mapped using SWISS-Pdb Viewer.
Plasmid construction and mutagenesis
Plasmids were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). Briefly, the full-length coding sequences of wild-type and mutant PADI6 (NM_207421.4) were synthesized and inserted into the p.cDNA3.1(+) vector with a C-terminal Flag tag. Sanger sequencing confirmed the wild-type and mutant clones.
Cell culture and transfection
Human embryonic kidney 293T (HEK293T) cells were kindly provided by the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries) supplemented with 10% fetal bovine serum (Biological Industries) and 1% penicillin/streptomycin (Gibco) in a humidified incubator with a 5% CO2 atmosphere at 37 °C. Transient transfections were performed using the Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. DNase and RNase water of equal volumes were added into the blank groups as controls.
The cells were washed thrice with cold PBS after transfection for 48 h. Cells were harvested and lysed with RIPA buffer (Beyotime Biotechnology) containing with 1× protease inhibitor cocktail (Beyotime Biotechnology). After incubating on the ice for 20 min and centrifuging at 12,000 rpm at 4 °C for 20 min, protein lysates were collected in a new centrifuge tube. After quantification using a bicinchoninic acid kit (Beyotime Biotechnology), equal amounts of samples were denatured in protein loading buffer (NCM Biotech) and heated at 100 °C for 10 min. Protein samples were separated by 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). The membranes were blocked in 5% skim milk diluted in 1× Tris-buffered saline with Tween 20 (TBST) for 1 h and incubated at 4 °C overnight with primary antibodies against Flag (1:1000, 8146S, Cell Signaling Technology) or GAPDH (1:10,000, 60004-1-Ig, Proteintech). After 1 h incubation with the corresponding secondary antibodies (1:10,000, 7076S, Cell Signaling Technology) for 1 h at room temperature on the secondary day, the membranes were visualized using Amersham ImageQuant 800 (Cytiva, United States) after detection with BeyoECL Plus (Beyotime).
After transfection for 48 h, cells on slides were fixed with 4% paraformaldehyde for 20 min, and then incubated in permeabilizing solution (0.02% Triton X-100 in PBS) for 15 min and blocking buffer (5% goat serum in PBS) for 1 h. Anti-Flag antibody (1:400, 8146S, Cell Signaling Technology) was added at 4 °C overnight. On the secondary day, the cells were incubated with CoraLite488-conjugated goat anti-mouse IgG(H+L) (1:400, SA00013-1, Proteintech) for 1 h to visualize PADI6-Flag staining. DAPI Fluoromount-G (SouthernBiotech) was used to label DNA. Finally, the cells were imaged on a fluorescence microscope (Leica, Germany).
RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from HEK293T cells 48 h after transfection using the Total RNA Purification Kit (SimGen). Reverse transcription and amplification were performed using the Evo M-MLV RT Mix Kit (Accurate Biotechnology), and RT-qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) according to the manufacturer’s instructions. All reactions were performed with at least three technical replicates. The relative expression level was calculated using 2−△△Ct normalized to endogenous GAPDH expression. Primers used in the experiments are listed in Additional file 1: Table S1.
All experiments were independently conducted in triplicate. RT-qPCR analysis was performed using Student’s t-tests when comparing experimental groups. Statistical analyses were performed using GraphPad Prism 8.0. P < 0.05 was considered as statistically significant.