Patients and ethical approval

Our previous study reported that 2 of 12 patients with HWWS harbored the CHD1L variant c.348-1G>C [6]. Therefore, an additional 85 Chinese Han patients affected by MDAs and diagnosed and treated at Beijing Obstetrics and Gynecology Hospital, Capital Medical University, from January 2019 to February 2024 were recruited. One hundred unrelated Chinese women with normal phenotypes confirmed by imaging examination and/or hysteroscopy were enrolled as controls. All individuals had 46,XX karyotypes. Two experienced gynecologists conducted separate evaluations of patients’ profiles for the purpose of the study, and agreements were reached. MDAs were discriminated by classification systems, including VCUAM [11], ESHRE/ESGE [12], and MAC2021 [13]. The clinical profiles of the patients are summarized in Table 1. Their body mass indices were within the normal range, their body hair was typically distributed, and the hormone analysis results remained normal. Fc-K-1 did not show any significant clinical symptoms and was diagnosed as a partial septate uterus with left renal agenesis at a check-up when she was 26 years old. Fc-U-46491 exhibited a complete septate uterus with duplicated cervices and a longitudinal vaginal septum. She underwent full-term delivery through a cesarean section owing to abnormalities in the birth canal, a typical complication associated with MDAs. Both patients denied a family history of urogenital abnormalities.

Table 1 The clinical profile of the patients with MDAs harboring CHD1L mutations

The research received approval from the Ethics Committees of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (2018-KY-027-01 and 2021-KY-032-01), following the ethical principles set forth in the Declaration of Helsinki.

WES, in silico analysis and Sanger sequencing

The genomic DNA from all recruited participants was isolated from peripheral blood using a TIANamp Blood DNA Kit (DP348; TIANGEN, China) according to established protocols. WES was carried out on Illumina NovaSeq 6000 sequencers, generating paired-end reads of 150 bp for each reaction. Following variant calling and annotation, cases with CHD1L variants in line with the specified criteria were included: (1) potentially affected protein sequences; (2) minor allele frequencies less than 0.01 in accordance with the Genome Aggregation Database; and (3) algorithm prediction of deleterious or likely deleterious [14]. More detailed information about the methodology was provided in a previous publication [6].

Sanger sequencing was employed to confirm the identified variants. The primer pairs used to verify the polymerase chain reaction (PCR) products are listed in Supplementary Table 1. Sequencing was performed in an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).

Molecular modeling and simulations of CHD1L proteins

The predicted structures of CHD1L and poly(ADP-ribosyl)ation polymerase 1 (PARP1) were obtained from the AlphaFold Protein Structure Database (AF-Q86WJ1-F1 and AF-P09874-F1, respectively) [15]. The mutant proteins were then generated using PyMOL by introducing corresponding modifications to the remaining sites in the initial framework. Subsequently, molecular dynamics simulations were conducted with Gromacs2022.3 software at a constant temperature of 298 K and an atmospheric pressure of 1 bar for 100 ns [16]. The force field AMBER14SB was used, with water molecules as the solvent (Tip3p water model) [17]. The simulation system utilized the steepest descent method for energy minimization and then proceeded to NVT and NPT equilibrium, with a 2 fs coupling constant and a 100 ps duration. The subsequent simulation lasted for 20 ns, during which 10,000,000 steps were carried out with a step length of 2 fs. The Particle Mesh Ewald method was utilized to handle long-range electrostatic interactions. The cutoff distances for calculating electrostatic and van der Waals interactions were set at 12 Å and 10 Å, respectively. Trajectory analysis was performed using the tool within the software. The execution of molecular docking between the CHD1L and PARP1 proteins was simulated in ZDOCK [18].

C57BL/6 mice

The C57BL/6 mice were kept in specific-pathogen-free animal facilities, where the temperature was maintained at 22–26 °C, with a 12-h light/dark cycle. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Beijing Obstetrics and Gynecology Hospital.

Plasmid construction and transfection

The overexpression plasmids pcDNA3.1(+)-CMV-human CHD1L-wild-type (WT)-3 × Flag (NM_004284) and pcDNA3.1(+)-CMV-human PARP1-HA (NM_001618) were purchased from YouBio Biology (China). Mutants of CHD1L related to the point of interest were generated from the WT plasmid. The empty vector pcDNA3.1(+) was used as the negative control. The procedures for constructing the plasmids for the minigene assay are detailed below. The pcDNA3.1(+)-GFP-WT/mutant CHD1L and pcDNA3.1(+)-mCherry-PARP1 plasmids were both created by adding N-terminal fluorophore sequences and eliminating C-terminal tagged protein sequences from the parent plasmids. Transient transfection was conducted using jetPRIME® Transfection Reagent (101000046; Polyplus, France) following the manufacturer’s instructions.

Cell culture and reagents

293FT cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (CT11995500BT; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (F8687; Sigma-Aldrich, USA), penicillin‒streptomycin (100×) (15070063; Gibco, USA), GlutaMAX™-I (35050079; Gibco, USA) and MEM NEAA (100×) (11140050; Gibco, USA). HeLa cells were cultivated in DMEM supplemented with 10% FBS and 1% penicillin‒streptomycin. The cells were incubated in a cell culture chamber at 37 °C with 5% CO2 in a humidified environment.

Antibodies

The related primary antibodies included GAPDH antibody (AC033; ABclonal, China), DYKDDDDK tag antibody (66008-4-Ig; ProteinTech, China), HA tag antibody (51064-2-AP; ProteinTech, China), anti-β actin antibody (TA-09; ZSGB-BIO, China), anti-CHD1L antibody (ab197019; Abcam, USA) and human PARP1 antibody (13371-1-AP; ProteinTech, China). The secondary antibodies used were anti-mouse/anti-rabbit IgG (H+L) biotinylated antibody (ZB-2305/ZB-2301; ZSGB-BIO, China) and anti-mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor™ 488 (A-11001; Invitrogen, USA). Antibodies were used with appropriate dilutions conforming to standard protocols.

Minigene splicing assay

It was hypothesized that the CHD1L variant c.348-1G>C might impact the splicing of exon 4; thus, vectors encompassing the WT or mutant fragments were constructed. PCR was used to amplify the sequences containing exon 3 (107 bp), intron 3 (836 bp), exon 4 (115 bp), intron 4 (603 bp), and exon 5 (32 bp) with the addition of ATG and TGA from the templates of the genomic DNA of Fc-H-5/Fc-H-8 and the control. The product was collected using an Agarose Gel DNA Purification and Recovery Kit (DH101; Biomed, China). A restriction enzyme sequence containing the KpnI (5′) and XhoI (3′) digestion sites flanking the whole sequence was subsequently inserted into the multicloning site of the pcDNA3.1(+) empty vector. Total RNA was isolated from the 293FT cells transfected with these plasmids using a HiPure Total RNA Mini Kit (R4111; Magen, China), and cDNA was synthesized by reverse transcription (RT) using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311; TransGen Biotech, China). The cDNA was subsequently amplified by PCR using the primers listed in Supplementary Table 2, which flanked the target minigene. The PCR products were separated by agarose gel electrophoresis and sequenced, and the transcripts of the WT and mutant fragments were identified.

RNA extraction and RT-qPCR

The methods used for RNA extraction and cDNA synthesis are described above. Quantitative real-time PCR (qPCR) was performed in triplicate with the PerfectStart Green qPCR Super Mix (AQ601; TransGen Biotech, China) on a fluorescence instrument (LightCycler 480 II; Roche, Basel, Switzerland). The relative gene expression levels were normalized to the critical threshold of the housekeeping gene ACTB. The primers used are listed in Supplementary Table 2.

Western immunoblotting

Total cell lysates from the transfected 293FT cells were prepared in RIPA buffer containing protease and phosphatase inhibitor cocktails (P0013B and P1046; Beyotime Biotechnology, China). The protein concentration was quantified using an Enhanced BCA Protein Assay Kit (P0009; Beyotime Biotechnology, China). Equal amounts of lysates were subjected to 8–12% SDS-PAGE and transferred onto a 0.22 μm polyvinylidene fluoride membranes (ISEQ00010; Merck Millipore, Ireland). The membranes were blocked in 5% nonfat milk in TBST (0.1% Tween-20 in Tris-buffered saline). After being probed with appropriate primary antibodies followed by secondary antibodies, the proteins were visualized using Immobilon Western HRP Substrate Luminol Reagent (WBKLS0500; Millipore, USA) and a ChemiDoc imaging system (Bio-Rad, USA).

Immunofluorescence microscopy

293FT cells were cultivated and transfected with the corresponding CHD1L constructs at the appropriate density. After 48 h, the cells were centrifuged at 400 rpm to adhere to microscope slides (188105; CITOTEST Scientific, China) using a Shandon Cytospin™ 4 cytocentrifuge (Thermo Scientific, USA). The samples were then fixed with 4% paraformaldehyde (P1110; Solarbio, China), permeabilized with 0.3% Triton X-100 in Tris-buffered saline and blocked with 5% bovine serum albumin buffer. The cells were subsequently stained with the primary anti-FLAG antibody and secondary antibody conjugated with Alexa Fluor® 488. After washing with TBST, a drop of VECTASHIELD® antifade mounting medium with DAPI (H1200; Vector Laboratories, USA) was used to stain the nuclei and mount the cells. The sealed coverslips were visualized with an EVOS imaging system (AMF7000; Invitrogen, USA).

Scratch-wound assay

HeLa cells were transfected with the corresponding plasmids and incubated to reach a confluence greater than 90%. Next, the medium was removed, and the surface of the inoculated cells was gently scraped with a 10 μl pipette tip. After washing with PBS (P1020; Solarbio, China), DMEM supplemented with 0.5% FBS was added. The scratches were photographed at 0, 12 and 24 h. The area in which the cells migrated during the observation period was measured with ImageJ software. The results are presented as the migration rate (%), which was calculated as the ratio of the area of cell migration at 12 or 24 h and the initial area at 0 h.

Transwell migration assay

HeLa cells were seeded, transfected with target vectors and cultivated for 48 h. The cells were then collected and resuspended in serum-free DMEM to an appropriate density. The upper and lower wells of the plate (3422; Corning, USA) were filled with cell suspension and 600 μl of complete medium, respectively. After incubation for 24 h, the cells that had not passed through the wells were carefully removed using a cotton swab. The cells in the lower chamber were then fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet solution (G1064; Solarbio, China) for 15 min, and washed with PBS. The cells were photographed and counted.

Coimmunoprecipitation (Co-IP) assay

293FT cells were seeded and transfected with empty vector or WT or mutant CHD1L plasmids with or without PARP1 constructs. After incubation, the cells were completely lysed with lysis buffer containing a protease inhibitor cocktail (P2181S; Beyotime, China) and centrifuged at 12,500×g for 10 min at 4 °C. Magnetic beads were conjugated with anti-FLAG, anti-HA and anti-PARP1 antibodies according to the instructions. Protein extracts were mixed with mouse IgG magnetic beads, which were used as a negative control, or magnetic beads (P2171 and P2108; Beyotime, China) with target antibodies at an appropriate ratio (25:1). The protein/bead mixtures were then incubated on a rotating wheel overnight at 4 °C. The beads were then rinsed with lysis buffer and resuspended in SDS-PAGE sample loading buffer. After denaturation at 98 °C for 5 min, the immunoprecipitated proteins were eluted and further evaluated by western immunoblotting.

Laser microirradiation

The experiment was conducted according to the thorough methodology elucidated in the published literature [19]. 293FT cells were cultured and cotransfected with GFP-tagged CHD1L plasmids and mCherry-tagged PARP1 plasmids. After 24 h, the cells were plated onto 35 mm glass-bottom dishes (801001; NEST, China) coated with poly-d-lysine. The culture medium was subsequently removed, and phenol red-free medium containing Hoechst 33342 (C1028; Beyotime, China) used for sensitization was added to the dishes. Laser microirradiation was carried out on an A1 HD25 confocal microscope (Nikon Instruments Inc., Japan) equipped with an incubation chamber for the regulation of humidity and CO2 levels with a 37 °C heating stage. A 405 nm laser, with a total power output of 405.7 mW, was used to induce DNA damage.

Statistics

The statistical analysis was performed in GraphPad Prism v9.4.1, and the details are specified in the relevant sections. A P value less than 0.05 was considered statistically significant. The error bars denote the standard deviation (SD).