A family with a proband diagnosed with male infertility was involved in this study. The family members who participated in this study were thoroughly informed about this study. This study was approved by the medical ethics committee of Tianjin Central Hospital of Gynecology Obstetrics (No. ZY2023001).

Genomic DNA (gDNA) was isolated from peripheral blood via the DNeasy Blood and Tissue Kit (QIAGEN, Germany). WES was performed via the sequencing platform of the Beijing Genomics Institute (BGI). All steps were performed according to the manufacturer’s instructions. Exomes were hybridized and captured by the xGen Exome Research Panel of Integrated DNA Technologies (IDT, America) and sequenced on the MGI-2000. The average sequencing depth was 100x‒150x, and the data quality was Q20 ≥ 90% and Q30 ≥ 90%. The original data obtained by sequencing were filtered through fastp software (Chen et al., 2018) and then passed through BWA software (Houtgast et al., 2018). The sequenced reads were collected, filtered for quality, and aligned to the human reference genome (hg19/GRCh37). The sequenced variants were annotated via ANNOVAR software (Wang et al., 2010)^, and mutations were screened on the basis of patients’ clinical information, population databases, disease databases, and bioinformatic prediction tools. Candidate pathogenic variants were scored in accordance with the criteria set by the American College of Medical Genetics and Genomics (ACMG) (Richards et al., 2015) and confirmed by Sanger sequencing.

To construct the mini gene, we amplified the SYCP2 fragment from the sectional intron26 (627 bp) to the sectional intron27 (84 bp) via nested polymerase chain reaction (PCR) with a primer pair and added the endonuclease recognition sequences of KpnI and XhoI to the front and end of the fragment, respectively. The detailed methods were presented in the supplementary materials. The PCR products were purified via alcohol and digested along with the vector pcMINI via the endonucleases KpnI and XhoI (New England Biolabs, America). The digested PCR products and vectors were purified via electrophoresis and ligated together with T4 ligase (New England Biolabs, America). Ligase products were transformed into DH5α competent cells, which were subsequently plated on LB plates coated with ampicillin. Single clones were then selected for proliferation and Sanger sequencing. The identified colonies were amplified, and plasmid DNA without endotoxin was extracted via the Rapid Plasmid Mini Kit (Simgen, China). The primers used in this study are listed in the Supplementary Material.

Human embryonic kidney 293T (HEK293T) cells and HeLa cells were cultured in DMEM/high glucose (Gibco, America) supplemented with 10% FBS (Sigma, America) in a 37°C constant-temperature water bath incubator at 5% CO₂. The cells were dissociated into single cells using trypsin-EDTA (Thermo Fisher, America) after they reached 80% confluence. The cells were counted, and 4 × 10⁵ cells were seeded in 6-well plates 24 h before transfection. The cell medium was changed to Opti-MEM (Thermo Fisher, America) 2 h before transfection. Lipofectamine 2000 (Thermo Fisher, America) was incubated for 20 min at room temperature, the mixture was mixed thoroughly with 1 μg of plasmid, and then, the mixture was gently added to the cell medium. The cell medium was replaced with fresh culture medium supplemented with 10% FBS 12 h after transfection.

Total RNA was extracted 48 h after transfection via TRIzol reagent (TaKaRa, China) following routine procedures. Reverse transcription‒polymerase chain reaction (RT-PCR) was performed via ABScript III RT Master Mix for qPCR with gDNA Remover (ABclonal, China) according to the manufacturer’s instructions. cDNA was used as a template for PCR with the primer pair F/R (Supplementary Material). The amplification products were subjected to electrophoresis on a 1% agarose gel and Sanger sequencing.

The SYCP2 gene sequence was obtained from the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene/). The molecular structure of the cryo-EM structure of GATOR1 was viewed with Mol* View (Sehnal et al., 2021) and stored in the RCSB PDB (Berman et al., 2000).

We sought to identify the genetic etiology of infertility for a male research participant who presented with a 2-year history of infertility at age 30. His assessment indicated asthenoteratozoospermia (AT) (the semen test results were shown in Table 1) in accordance with the WHO Laboratory Manual for the Examination and Handling of Human Semen, fifth edition. The patient displayed no dysmorphic features and had normal serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Y chromosome microdeletions were normal. The couple pursued ICSI as a treatment for male infertility. However, the couple did not become pregnant after ICSI treatment at the center. Despite multiple attempts at ICSI and despite successful fertilization, the couple was unable to obtain viable embryos. Eventually, the patient discontinued the treatment.

To explore the genetic factors contributing to the infertility of the patient, WES and subsequent validation through Sanger sequencing were performed. The patient was found to have a heterozygous variant in intron 27 of SYCP2, NM_014258.4:c.2600 + 5G>C (Figure 1A). Sanger sequencing via the primer SYCP2-F/R confirmed that this variant was inherited from the patient’s mother, who had a normal phenotype (Figure 1B).

Notably, this variant was absent in several major population databases, including the 1KGP (1000 Genomes Project, Phase 3), ESP6500 (Genome Aggregation Database, V2), gnomAD (Genome Aggregation Database, r2.0.1) and ExAC (Exome Aggregation Consortium, r0.3.1) databases, highlighting its rarity within these populations. This is the first report of this phenomenon in this study. Bioinformatics analysis via “Ada” and “RF” scores predicted that this variant was likely to influence splicing (Supplementary Table S2). According to the ACMG guidelines, the novel variation c.2600 + 5G>C was designated as a variant of uncertain significance (VUS).

To validate the effect of the c.2600 + 5G>C variation on RNA alternative splicing, we conducted a mine gene splicing assay by constructing containing the wild-type (wt) and mutation-type (mut) target DNA fragments (Figure 2A). The vectors were confirmed by Sanger sequencing (Figure 2B). Plasmid DNA without endotoxin was transfected into HEK293T cells and HeLa cells. The total RNA of the transfected cells was extracted, and reverse transcription was performed to obtain cDNA. Agarose electrophoresis of the RT-PCR products revealed two distinct splicing patterns (Figures 2C,D). The full uncropped Gels image presented in Supplementary Figure S2. Sanger sequencing revealed that abnormal splicing occurred in cells transfected with the mutation plasmid and that the mutation C.2600 + 5G>C affected the normal splicing of gene mRNA (Figure 2E). The detection results of pcMINI and PCMINI-C were consistent, as shown inSupplementary Figure S1. There was one abnormal transcript after mutation: exon 27 skipping. The mutation caused exon 27 skipping, which was expressed in the cDNA as c.2530_2600del. Exon 27 skipping caused a subsequent frameshift and produced an early stop codon in exon 28, which might produce a truncated protein 844 aa in length. Thus, the variation was described as SYCP2:c.2600 + 5G>C (p.Lys845*).

We further analysed the cDNA sequence of SYCP2 and found that the variation led to a frameshift and early appearance of the stop codon, which resulted in the prematuration of SYCP2 with 844 amino acid residues. Mutation of SYCP2 resulted in the loss of the whole coiled-coil (CC) domain (Figure 3). In rodents, SYCP2 directly interacts with SYCP1 and SYCP3 through its C-terminal domain and internal curly helix domain, respectively (Winkel et al., 2009). The deletion of the SYCP3-interacting domain of SYCP2 leads to severe defects in SC formation in mice, and males are sterile (Yang et al., 2006). The deletion of the CC domain meant that the protein was fundamentally changed and that its function was severely impaired.