In April 2024, a 22-year-old woman with intellectual disability visited the genetic counseling clinic at our hospital, accompanied by her family. We conducted an extensive clinical survey and then collected peripheral blood samples from the family members in the pedigree for genetic testing.

The extracted peripheral blood DNA was digested, fragmented, and end-repaired. The exons and adjacent splicing regions of the genes were captured using Amcare probes (AmCare Genomics Lab, Guangzhou, China). A DNA library was then prepared using the Gene Sequencing Library Kit (AmCare Genomics Lab, Guangzhou, China) and subjected to high-throughput sequencing on the AmCareSeq-2000 sequencer (AmCare Genomics Lab, Guangzhou, China).

The obtained next-generation sequencing (NGS) data were processed sequentially with the fastp, BWA, GATK, and ANNOVAR for data cleaning, alignment to the hg19 genome, variant calling, and variant annotation. Variant analysis and interpretation incorporated pathogenic variant databases (ClinVar, HGMD, DECIPHER, ISCA, and NCBI), population databases (gnomAD, ExAC), and the OMIM database. Variant filtering was performed using the following criteria: (1) Low-quality (read depth <20×, an allele fraction <30%). (2) Variants with a minor allele frequency ≥0.005, based on gnomAD and ExAC. (3) Variants located outside of coding regions and splicing regions (defined as the eight bases flanking each exon–intron boundary) were excluded. (4) Synonymous variants within the exome were removed. Given the clear autosomal dominant inheritance pattern revealed by pedigree analysis, we focused on variants inherited from the mother. The pathogenicity of variants was classified based on ACMG variant classification guidelines and supplemental guidelines, combined with clinical manifestations and examination results (Richards et al., 2015).

Exome sequencing results were validated using Sanger sequencing. Primers for candidate variants were designed with Primer Premier v5.0, followed by PCR amplification and purification. Sanger sequencing was performed on an ABI Prism 3700 sequencer.

The 3D structure of AGO2 was predicted and modeled using the AlphaFold program. The p.Cys717Arg and p. Gly733Arg variants was introduced into the wild-type AGO2 protein model using Swiss-Pdb Viewer, followed by energy minimization. Molecular dynamics simulations were performed using the GROMACS (version 2020.6) (Rakhshani et al., 2019). The AGO2-WT, AGO2-(p.Cys717Arg), and AGO2-(p. Gly733Arg) model were simulated for 60 ns in the CHARMM36 force field (Soteras Gutierrez et al., 2016). The wild-type and variant protein structures were solvated in a triclinic water box, maintaining a minimum buffer distance of 1.0 nm between the protein surfaces and the edges of the simulation cell. The system was neutralized by adding Na⁺ and Cl⁻ ions to achieve charge balance. Subsequent energy minimization was conducted using the steepest descent algorithm to eliminate steric clashes and unfavorable geometries. Finally, the system underwent equilibration in the NVT ensemble at 300 K for 120 ns to ensure thermal and conformational stability prior to production simulations. After completing the molecular dynamics simulation, the following commands: gmx rms, gmx rmsf, gmx gyrate, gmx sasa, gmx hbond, and gmx do_dssp were used to calculate the following properties for both the wild-type and variant proteins: root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), the number of intra-protein hydrogen bonds, and changes in secondary structure.

The evolutionary conservation of amino acid (AA) residues affected by specific missense variants was analyzed using UGENE (http://ugene.net/) with default parameters.

This study was reviewed and approved in advance by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (approval No. 2025-KY-029-01). All procedures involving human participants adhered to the Declaration of Helsinki 1964 and its subsequent revisions, or other applicable ethical standards.

All participants in this study were thoroughly informed about the study’s purpose, procedures, potential risks, and benefits. They were assured of their right to withdraw from the study at any time without facing any consequences. Written informed consent was obtained from all patients or their legal representatives, covering both genetic testing and the publication of the findings.

The proband is a 22-year-old female, with primary symptoms including intellectual disability, motor developmental delay, impaired speech development, impaired receptive language, gait abnormalities, attention deficit hyperactivity disorder, visual impairment, open mouth appearance, low nasal bridge, small nose, and lower eyebrow tail. The family includes four affected individuals: the proband (IV4), along with II4, III1, and III3. The pedigree is shown in Figure 1A. These four affected individuals in the family exhibit similar symptoms. All four affected individuals exhibit comparable clinical features, as detailed in Supplementary Table S1.

First, whole-exome sequencing (WES) was performed on the proband (IV4) and her parents (III2, III3). The results showed that both the proband (IV4) and her mother (III3) carried the AGO2 (NM_012154.5): c.2149T>C (p.Cys717Arg) variant, which was confirmed by Sanger sequencing, as shown in Figure 1B. Additional variants identified by Trio-WES included the following: 1) ALDH18A1 (NM_002860.4): c.683C>T (p.P228L), a de novo variant classified as VUS (PM2_P + PP3). 2) ASH1L (NM_018489.3): c.6238G>A (p.V2080I), a paternally inherited variant, also classified as VUS (PM2_P + PP3). 3) TUBGCP2 (NM_006659.4): compound heterozygous variants c.854A>G (p.Y285C), inherited from the mother and classified as VUS (PM2_P + PP3), and c.373G>A (p.A125T), inherited from the father and likewise classified as VUS (PM2_P + PP3). Taking into account the mode of inheritance and the clinical phenotype, we preliminarily consider the AGO2 (NM_012154.5): c.2149T>C (p.Cys717Arg) variant to be the likely cause of the proband’s intellectual disability. To further confirm the pathogenicity of this variant, peripheral blood was collected from all family members, and Sanger sequencing was used to check whether they carried the above variant. The Sanger sequencing results showed that all the proband (IV4), her mother (III3), her uncle (III1), and her grandmother (II4) carried the variant, while other intellectually normal family members did not carry the variant. The proband’s grandmother (II4) carried a de novo variant. The status of symptoms and variant in this family are shown in Figure 1A.

Next, we assessed the pathogenicity of the AGO2 (NM_012154.5): c.2149T>C (p.Cys717Arg) variant according to the ACMG guidelines. The pathogenicity rating was classified as Likely Pathogenic (PM2_P+PP2+PS2_M+PP1+PP3). The evidence items are as follows: 1) PM2_P: The variant was not found in the gnomAD database. 2) PP2: The missense Z score in the gnomAD database is 6.06, which is greater than 3.09.3) PS2_M: Individual II4 carries a verified de novo variant, and her symptoms are consistent with, but not specific to LESKRES syndrome. 4) PP1: The inheritance pattern of AGO2 is autosomal dominant, with three affected segregations, yielding an LOD score of 0.9, which is greater than 0.6.5) PP3: Multiple prediction tools suggest that this variant is damaging (SIFT, Polyphen2_HVAR, LRT, MutationTaster, PROVEAN, VEST3, M_CAP, CADD, DANN, FATHMM_MKL, Eigen, GenoCanyon, fitCons_pred, ReVe, ClinPred_pred, REVEL). We also analyzed the conservation of the amino acid residue at position 717, and the results showed that the amino acid at position 717 is highly conserved across species (Figure 1C). The amino acid at position 717 is predicted to be conserved by multiple software tools (GERP, phyloP, phastCons, SiPhy_pred).

The 3D structures of wild-type AGO2 protein and the Cys717Arg variant are shown in Figures 2A,B. Amino acid residues 517–818 (shown in brown) represent the Piwi domain of the AGO2 protein. In the wild-type protein, the Cys residue at position 717 does not form hydrogen bonds with other amino acid residues (Figure 2A). However, in the Cys717Arg variant, the Arg residue at position 717 forms a hydrogen bond with the Glu residue at position 722 (Figure 2B). In the 60 ns molecular dynamics simulation, the Gly733Arg variant was used as a positive control. Compared to the wild-type protein, the number of hydrogen bonds formed between the amino acid residue at position 717 and other residues in the protein was markedly increased in the Cys717Arg variant protein (Figure 2G). Similarly, in the positive control Gly733Arg variant protein, the number of hydrogen bonds formed between the residue at position 733 and other residues within the protein was also significantly increased (Figure 2H). Additionally, the SASA of the Cys717Arg and Gly733Arg variant was significantly greater than that of the wild-type protein (Figure 2F). The RMSD, RMSF and gyrate of the Cys717Arg and Gly733Arg variants showed no significant changes (Figures 2C-E).

This study summarized the pathogenic AGO2 variants reported to date, as illustrated in Figure 3. Including the variant identified in this study, a total of 14 pathogenic variants have been reported. Notably, the chr8.hg19:g. (141,582,269–141,817,600) del variant is not shown in Figure 3. With the exception of p.Phe182del, all identified variants are missense. Analysis of the distribution of pathogenic variants in the AGO2 gene reveals three hotspot regions at amino acid residues 182–203, 357–367, and 717–760. The p.Cys717Arg variant identified in this study is located within the 717–760 region, which corresponds to the Piwi domain.

Building upon the work of Lessel et al., this study also summarizes the clinical manifestations associated with AGO2 variants, as detailed in Supplementary Table S1. Phenotypic analysis of affected individuals indicates that intellectual disability (100%, 25/25), motor developmental delay (100%, 25/25), delayed independent walking (100% > 15 months, 22/22), impaired speech development (100%, 25/25), and impaired receptive language (100%, 17/17) are the most frequently clinical features. Seizures (67%, 8/22), epicanthic folds (64%, 16/25), gait abnormalities (59%, 9/18), attention deficit hyperactivity disorder (63%, 12/19), Abnormality of brain morphology (53%, 9/17), and open mouth appearance (52%, 13/25) are the secondly frequently clinical features.