Study Participants
We recruited a Chinese pedigree with two siblings with CBAVD from Henan Provincial People's Hospital. The proband was 31 years old and infertile for 2 years after marriage. Physical examination found bilateral absence of the vas deferens, and he was diagnosed with CBAVD at Henan Provincial People’s Hospital (Henan University People's Hospital) in combination with auxiliary examinations. Written informed consent was obtained from all participants. This study was approved by the Ethics Committee of the Henan Provincial People's Hospital.
Whole-exome sequencing
Genomic DNA was extracted from blood samples using the DNeasy Blood & Tissue Kit (Qiagen). IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies) was used for whole-exome sequencing of samples. A Qubit 2.0 fluorometer (Thermo Fisher Scientific) and 2100 Bioanalyzer high-sensitivity DNA assay (Agilent Technologies) were used to evaluate and measure the quantity and quality of sequencing libraries.
Qualified libraries were sequenced on the Illumina NovaSeq platform (Illumina, San Diego, USA) for 2 × 150-bp paired-end next-generation sequencing. BWA v0.7.13 was used to compare the FASTQ files with the human reference genome (hg19/GRCh37) [13]. Variants (single-nucleotide variants and indels) were genotyped from recalibrated BAM files using GATK 4.0 and annotated with ANNOVAR based on data such as HGVS variant description, population frequency, disease or phenotype, and variant function prediction [14, 15]. The American College of Medical Genetics (ACMG) guidelines classify variant types as pathogenic, probably pathogenic, variants of unknown significance (VUS), probably benign, or benign [16]. Copy number variants were called by the DNA copy R package and manually examined using the Integrative Genomics Viewer after filtering and classification according to the ACMG guidelines [17–19].
Sanger sequencing
Sanger sequencing was used to confirm mutations and analyze co-segregation using the following primers: forward 5′-F-TTCCTGCATCCATGGCATCT-3′ and reverse 5’-GCCAACTCCACCGTCAACAG-3’ for c.2144delA, and forward 5’-ATGTCCTCTAGAAACCGTATGC-3′ and reverse 5′-CCAAAAATACCTTCCAGCACTA-3′ for c.1210-11T > G.
Plasmid construction
RNA was extracted from healthy human blood samples using the RNAsimple Total RNA Kit (DP419, TIANGEN), and reverse transcription was performed using First-Strand cDNA Synthesis SuperMix (AT301, TransGen Biotech) to synthesize cDNA. The target fragment was amplified with DNA polymerase (TaKaRa Premix Taq Version 2.0 plus dye) using the following primers (designed by Primer 5.0 software): 5'-ATGCAGAGGTCGCCTCTGG-3' and 5'-CAACCAAAGAAGCAGCCACC-3'. The amplification products were cloned into the polyclonal site of the pEGFP-C1 vector using the restriction endonucleases BamHI and EcoRI (New England Biolabs). The wild-type plasmid was then extracted using the TIANprep Mini Plasmid Kit (DP103, TIANGEN) and sent to Zhengzhou Sunya Biotechnology Co., Ltd. for Sanger sequencing verification.
Mutant plasmid construction and identification
Mutant plasmids were constructed using the Mut Express II Fast Mutagenesis Kit V2 (C214, Vazyme). Using the wild-type plasmid as a template, the following primers were designed and used: 5'-CCATTGTGCAAAGACTCCCTTACAAATGAATGGC-3' and 5'-GGAGTCTTTGCACAATGGAAAATTTTCGTATAGA-3'. The primer annealing temperature was set at 63°C. The cyclized recombinant products were transformed into DH5α cells (C502, Vazyme), cultured and identified by PCR. Next, the positive clones were screened, and the plasmids were extracted using a TIANprep Mini Plasmid Kit (DP103, TIANGEN) to obtain the mutant plasmids, which were sent to Zhengzhou Sunya Biotechnology Co., Ltd. for Sanger sequencing to verify whether the targeted mutation was successfully constructed.
Cell culture and transient transfection assays
HEK293T cells frozen in liquid nitrogen were resuscitated according to the conventional method, DMEM culture medium with a volume fraction of 10% fetal bovine serum was added, and the cells were incubated in an incubator at 37°C with 5% CO2. Cells were transfected in 6-well plates when the cell density reached 80%-90% and then transfected using the Lipofectamine 2000 DNA Transfection Reagent Protocol (Life Technologies) when the cell density reached 80%-90% again. Lipofectamine 2000 reagent and plasmids were diluted with OPTI-MEM (Gibco) and then mixed together. The mixture was incubated for 20 min at room temperature and then added dropwise to HEK293T cell culture medium in a 6-well plate, which was shaken gently before placing it in the incubator. The expression of green fluorescent protein was observed by inverted fluorescence microscopy after 48 hours of transfection.
Western blot
After transfection for 48 hours, cells were washed with PBS and lysed with RIPA Lysis Buffer (P0013B, Beyotime Biotechnology) containing protease inhibitors. SDS‒PAGE Sample Loading Buffer 6X (Beyotime Biotechnology) was added to the cell samples, and proteins were denatured by boiling at high temperature. After the samples were cooled, SDS‒PAGE protein gel electrophoresis was performed. A 6% SDS‒PAGE gel (P0012A, Beyotime Biotechnology) was prepared, and the extracted protein samples were separated by gel electrophoresis, transferred to PVDF membranes, blocked with 10% skimmed milk powder for 2 hours at room temperature, washed with TBST, and then incubated overnight at 4°C with GFP primary antibody (50430, Proteintech) diluted 1:2000. The membrane was washed with TBST, and horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (A0208, Beyotime Biotechnology) diluted 1:1000 was added and incubated for 2 hours at room temperature. Finally, the membrane was exposed to ultrasensitive ECL reagent (P0018, Beyotime Biotechnology) for color development, and the protein bands were imaged by a high-sensitivity chemiluminescence ChemiDoc XRS system (Bio-Rad).