The patient was a 3-year-old boy who first presented at the age of 2 years for evaluation of developmental delay. There was no family history of congenital anomalies or intellectual disability/neurodevelopmental disorders (NDD). He was born at 38 weeks’ gestation with normal birth weight and length following an uncomplicated pregnancy. His developmental milestones were delayed; he raised his head at 5 months, sat independently at 10 months, stood up at 18 months and walked at 19 months. The boy was followed up until the age of 3 years and 10 months. At the last follow-up, he still could not say any words. His height was 92 cm (<10th centile) and weight was 12 kg (<10th centile). His occipitofrontal circumference was within the normal range. The electrocardiogram and cerebral magnetic resonance imaging were normal. His vision and hearing evaluations were both normal. Facial dysmorphisms, limb anomalies, autistic features and other behavioral anomalies have not been observed.

Trio-based whole-exome sequencing (trio-WES) was performed on genomic DNA (gDNA) of peripheral blood samples from this boy and his patients using the Blood Genome Column Medium Extraction Kit (Kangweishiji, China) according to the manufacturer's instructions. The extracted DNA samples were subjected to quality control using a Qubit 2.0 fluorimeter and electrophoresis with a 0.8% agarose gel for further protocols. The xGen™ Exome Research Panel v2 (designed by Integrated DNA Technologies) was used for WES. Quality control (QC) of the DNA library was performed using an Agilent 2100 Bioanalyzer System (Agilent, USA). DNA nanoball (DNB) preps of clinical samples were sequenced on ultra high throughput DNBSEQ-T7 platform (MGI, Shenzhen, China) with paired-end 150 nt strategy following manufacturer's protocol.

Sequencing data was analyzed according to our in-house (Chigene Translational Medicine Research Center) procedures. Raw data were processed quickly for adapter removal and low-quality read filtering, and then data quantity and data quality was statistics. The trimmed reads were then mapped to the University of California at Santa Cruz (UCSC) GRCh37/hg19 reference genome using Burrows-Wheeler Aligner (BWA) software (version 0.7.17). The Genome Analysis ToolKit (GATK) software (version 4.2.1.0) was used for single nucleotide polymorphisms/variants (SNPs/SNVs) and short insertions/deletions (indels) (<50 bp) calling. Samtools (version 1.22.1) and Picard software (version 2.22.1) packages were used to generate clean binary alignment map (BAM) data by removing duplicate data. Variants were annotated for analysis using the single nucleotide polymorphism database (dbSNP, version 5.3), gnomAD exomes database (version 4.1.0) and Chigene in-house minor allele frequency (MAF) database. Tools of pathogenicity prediction like REVELAL and AlphaMissense were used for predicting possible impact of variants. As a prioritized pathogenicity annotation to the American College of Medical Genetics and Genomics (ACMG) guidelines, Online Mendelian Inheritance in Man (OMIM), Human Gene Mutation Database (HGMD) and ClinVar databases were used as conferences of pathogenicity of every variant.

As per the guidelines of ACMG for interpreting sequence variants, variants were classified. Classification considered the position of the variant in the human genome, MAF, the pathogenicity prediction of variants, disease mechanism, clinical phenotypes, literature evidence, evolutionary conservation.

gDNA samples was used for the verification of this variant. All reactions were carried out in a total volume of 20 μl using the Taq DNA polymerase. After confirmation of the size of the amplicons, the PCR products were purified by standard protocol and Sanger sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI 3730 Applied Biosystem, USA) for the verification of candidate variants. The STAG1 gene primers used for PCR amplification were as follows: forward 5′-TTTATCCAGTGTTCAGGA-3′, reverse 5′-AGGGTACTTGTATGCCTAA-3′. The identification of variants was performed using Chromas software (version2.2.6, Technelysium, Australia).

Mutant STAG1 gene pcDNA3.1(+)-FLAG-STAG1-mut and control pcDNA3.1(+)-FLAG-STAG1-WT mammalian expression vectors were constructed and purchased from Wuhan Biorun Biosciences Co., Ltd. Human embryonic kidney (HEK)-293T cells were transfected with expression vectors in two tubes (one for the mutant expression vectors and one for the wild-type expression vectors). The constructed expression vectors were verified using Sanger sequencing. Then, Six hours after the transfection, we gently removed the lipofectamine-containing medium, replaced the medium with Dulbecco's modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS), and incubated the slide at 37 ℃ in a 5% CO₂ incubator until day 2. On day 3, the samples were collected and used in a reverse transcription-polymerase chain reaction (RT-PCR) to quantify the STAG1 mRNA expression and a Western blot (WB) assay to quantify the STAG1 protein expression. Each cell line underwent three independent replicates of the RT-PCR assay. The RT-PCR and Western blotting experiments were conducted in accordance with the manufacturer's instructions.

After transfecting HEK293T cells with wild-type (STAG1-WT) and mutant (STAG1-mut) expression vectors for 24 h, we centrifuged the transfected cells to collect them, and then extract the total RNA from the cells using the Trizol method. Total RNA concentration and quality were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized using NovoScript® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein, China) following the manufacturer's instructions. The cDNA concentration was about 1,200 ng/μl. The total reaction system was 20 µl, and the total RNA template was 500 ng. Quantitative PCR was performed using the Maxima SYBR Green/ROX qPCR Master Mix (2×) (Thermo Fisher Scientific, USA). Relative STAG1 mRNA expression levels were determined using 2^−ΔΔCt method, and the GAPDH gene was used as the reference gene. The qRT-PCR reaction was performed in triplicate, and mean values and standard deviations were calculated from the obtained data. The primers used for PCR amplification of STAG1 gene were as follows: forward 5′-AGAATTTGATGAGGACAGTGGTGA-3′, reverse 5′-TCAGGACTCCAATAAATTCACAAAA-3′.

After transfecting HEK293T cells with wild-type (STAG1-WT) and mutant (STAG1-mut) expression vectors for 24 h, western blot was performed using standard protocol. Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in a tris buffered saline with tween-20 (TBST) solution for 2 h at room temperature. Anti-Flag (at a dilution of 1:1,000) and anti-glyceraldehyde phosphate dehydrogenase (anti-GAPDH) dilutions (at a dilution of 1:1,000) were added respectively and incubated at 4 ℃ overnight. Horseradish peroxidase (HRP)-labeled secondary antibodies were added and incubated at room temperature for 1 h. GAPDH served as the loading control and internal reference protein. The loading amount of WB protein was 10 μg/well. Blot bands were quantified by densitometry using ImageJ software (ImageJ2). Protein levels were normalized to GAPDH. The relative STAG1 protein expression level is quantified by the fold change (FC) in the ratio of the grayscale between the STAG1 protein band and the internal reference protein band.

All data statistical comparisons between two groups (STAG1-WT and STAG1-mut) were evaluated by Student's t-test using Prism software (GraphPad Prism 10.5.0). P < 0.05 was considered statistically significant.

We searched the PubMed database using “STAG1 gene” as keywords. The search time was from the establishment of the databases to February 31, 2025. We reviewed STAG1-related cases with availability of clinical data including relevant genetic testing results, prenatal/postnatal manifestations and pregnancy outcomes.

Trio-WES identified a novel heterozygous frameshift variant in the STAG1 gene (NM_005862.3) c.500dup (p.Gly168TrpfsTer13). This variant was further verified by Sanger sequencing, confirming that it was a de novo variant (Figure 1). The allele frequency of this heterozygous variant has not been registered in population databases (1,000 Genomes Project, gnomAD, and dbSNP) or reported in disease databases (ClinVar, HGMD, OMIM) (PM2_Supporting). This patient was the only de novo occurrence of this variant (PS2_Supporting). The probability of being LoF intolerant (pLI) value of STAG1 gene in the gnomAD v4.1.0 was 1.0, which indicated that STAG1 gene was extremely intolerant to loss-of-function variants (pLI > 0.9). The frameshift STAG1 gene variant was a presumed LoF variant, LoF was a putative mechanism of STAG1-related disease (PVS1). As per the interpretation guidelines by ACMG (2), this novel variant was classified as “likely pathogenic”. We have submitted this variant to a public database [Leiden Open Variation Database (LOVD)], and the accession number can be found at the following URL: https://databases.lovd.nl/shared/individuals/00464708.

To evaluate the effect of the frameshift STAG1 variant, our functional studies revealed that this STAG1 variant, c.500dup, markedly decreased mRNA expression of STAG1 gene (Figure 2), leading to a lack of normal STAG1 protein (Figures 3, 4), compared to the STAG1-WT cell lines.