Mechanism of Ligand Binding to Theophylline RNA Aptamer.

Studying RNA-ligand interactions and quantifying their binding thermodynamics and kinetics are of particular relevance in the field of drug discovery. Here, we combined biochemical binding assays and accelerated molecular simulations to investigate ligand binding and dissociation in RNA using the theophylline-binding RNA as a model system. All-atom simulations using a Ligand Gaussian accelerated Molecular Dynamics method (LiGaMD) have captured repetitive binding and dissociation of theophylline and caffeine to RNA. Theophylline's binding free energy and kinetic rate constants align with our experimental data, while caffeine's binding affinity is over 10,000 times weaker, and its kinetics could not be determined. LiGaMD simulations allowed us to identify distinct low-energy conformations and multiple ligand binding pathways to RNA. Simulations revealed a "conformational selection" mechanism for ligand binding to the flexible RNA aptamer, which provides important mechanistic insights into ligand binding to the theophylline-binding model. Our findings suggest that compound docking using a structural ensemble of representative RNA conformations would be necessary for structure-based drug design of flexible RNA.

Synaptic Loss in Spinocerebellar Ataxia Type 3 Revealed by SV2A Positron Emission Tomography.

BACKGROUND: Severe reduced synaptic density was observed in spinocerebellar ataxia (SCA) in postmortem neuropathology, but in vivo assessment of synaptic loss remains challenging. OBJECTIVE SPINOCEREBELLAR ATAXIA TYPE 3: The objective of this study was to assess in vivo synaptic loss and its clinical correlates in spinocerebellar ataxia type 3 (SCA3) patients by synaptic vesicle glycoprotein 2A (SV2A)-positron emission tomography (PET) imaging. METHODS: We recruited 74 SCA3 individuals including preataxic and ataxic stages and divided into two cohorts. All participants received SV2A-PET imaging using 18 F-SynVesT-1 for synaptic density assessment. Specifically, cohort 1 received standard PET procedure and quantified neurofilament light chain (NfL), and cohort 2 received simplified PET procedure for exploratory purpose. Bivariate correlation was performed between synaptic loss and clinical as well as genetic assessments. RESULTS: In cohort 1, significant reductions of synaptic density were observed in cerebellum and brainstem in SCA3 ataxia stage compared to preataxic stage and controls. Vermis was found significantly involved in preataxic stage compared to controls. Receiver operating characteristic (ROC) curves highlighted SV2A of vermis, pons, and medulla differentiating preataxic stage from ataxic stage, and SV2A combined with NfL improved the performance. Synaptic density was significantly negatively correlated with disease severity in cerebellum and brainstem (International Co-operative Ataxia Rating Scale: ρ ranging from -0.467 to -0.667, P ≤ 0.002; Scale of Assessment and Rating of Ataxia: ρ ranging from -0.465 to -0.586, P ≤ 0.002). SV2A reduction tendency of cerebellum and brainstem identified in cohort 1 was observed in cohort 2 with simplified PET procedure. CONCLUSIONS: We first identified in vivo synaptic loss was related to disease severity of SCA3, suggesting SV2A PET could be a promising clinical biomarker for disease progression of SCA3. © 2023 International Parkinson and Movement Disorder Society.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a ß-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of ß-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.

Biallelic Intronic AAGGG Expansion of RFC1 is Related to Multiple System Atrophy.

OBJECTIVE: A recessive biallelic repeat expansion, (AAGGG)exp , in the RFC1 gene has been reported to be a frequent cause of late-onset ataxia. For cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), the recessive biallelic (AAGGG)exp genotype was present in ~92% of cases. This study aimed to examine whether the pentanucleotide repeat (PNR) was related to multiple system atrophy (MSA), which shares a spectrum of symptoms with CANVAS. METHODS: In this study, we screened the pathogenic (AAGGG)exp repeat and 5 other PNRs in 104 Chinese sporadic adult-onset ataxia of unknown aetiology (SAOA) patients, 282 MSA patients, and 203 unaffected individuals. Multiple molecular genetic tests were used, including long-range polymerase chain reaction (PCR), repeat-primed PCR (RP-PCR), Sanger sequencing, and Southern blot. Comprehensive clinical assessments were conducted, including neurological examination, neuroimaging, nerve electrophysiology, and examination of vestibular function. RESULTS: We identified biallelic (AAGGG)exp in 1 SAOA patient and 3 MSA patients. Additionally, 1 MSA patient had the (AAGGG)exp /(AAAGG)exp genotype with uncertain pathogenicity. We also described the carrier frequency for different PNRs in our cohorts. Furthermore, we summarized the distinct phenotypes of affected patients, suggesting that biallelic (AAGGG)exp in RFC1 could be associated with MSA and should be screened routinely in the MSA diagnostic workflow. INTERPRETATION: Our results expanded the clinical phenotypic spectrum of RFC1-related disorders and raised the possibility that MSA might share the same genetic background as CANVAS, which is crucial for re-evaluating the current CANVAS and MSA diagnostic criteria. ANN NEUROL 2020;88:1132-1143.

Prediction of the Age at Onset of Spinocerebellar Ataxia Type 3 with Machine Learning.

BACKGROUND: In polyglutamine (polyQ) disease, the investigation of the prediction of a patient's age at onset (AAO) facilitates the development of disease-modifying intervention and underpins the delay of disease onset and progression. Few polyQ disease studies have evaluated AAO predicted by machine-learning algorithms and linear regression methods. OBJECTIVE: The objective of this study was to develop a machine-learning model for AAO prediction in the largest spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) population from mainland China. METHODS: In this observational study, we introduced an innovative approach by systematically comparing the performance of 7 machine-learning algorithms with linear regression to explore AAO prediction in SCA3/MJD using CAG expansions of 10 polyQ-related genes, sex, and parental origin. RESULTS: Similar prediction performance of testing set and training set in each models were identified and few overfitting of training data was observed. Overall, the machine-learning-based XGBoost model exhibited the most favorable performance in AAO prediction over the traditional linear regression method and other 6 machine-learning algorithms for the training set and testing set. The optimal XGBoost model achieved mean absolute error, root mean square error, and median absolute error of 5.56, 7.13, 4.15 years, respectively, in testing set 1, with mean absolute error (4.78 years), root mean square error (6.31 years), and median absolute error (3.59 years) in testing set 2. CONCLUSION: Machine-learning algorithms can be used to predict AAO in patients with SCA3/MJD. The optimal XGBoost algorithm can provide a good reference for the establishment and optimization of prediction models for SCA3/MJD or other polyQ diseases. © 2020 International Parkinson and Movement Disorder Society.

RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays.

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered "undruggable" genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.

Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents.

Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide. Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as anti-breast cancer drugs.

Design, synthesis and biological evaluation of novel 3-substituted 4-anilino-coumarin derivatives as antitumor agents.

Various 3-substituted 4-anilino-coumarin derivatives have been designed, synthesized and their anti-proliferative properties have been studied. The in vitro cytotoxicity screening was performed against MCF-7, HepG2, HCT116 and Panc-1 cancer cell lines by MTT assay. Most of the synthesized compounds exhibited comparable anti-proliferative activity to the positive control 5-Fluorouracil against these four tested cancer cell lines. Among the different substituents at C-3 position of coumarin scaffold, 3-trifluoroacetyl group showed the most promising results. Especially, compounds 33d (IC50=16.57, 5.45, 4.42 and 5.16µM) and 33e (IC50=20.14, 6.71, 4.62 and 5.62µM) showed excellent anti-proliferative activities on MCF-7, HepG2, HCT116 and Panc-1 cell lines respectively. In addition, cell cycle analysis and apoptosis activation revealed that 33d induced G2/M phase arrest and apoptosis in MCF-7 cells in a dose-dependent manner. Low toxicity of compounds 33d and 33e was observed against human umbilical vein endothelial cells (HUVECs), suggesting their acceptable safety profiles in normal cells. Furthermore, the results of in silico ADME studies indicated that both 33d and 33e exhibited good pharmacokinetic properties.

Synthesis and biological evaluation of 2,3-diaryl isoquinolinone derivatives as anti-breast cancer agents targeting ERα and VEGFR-2.

The estrogen receptor α is recognized as important pharmaceutical target for breast cancer therapy, and vascular endothelial growth factor receptors (VEGFRs) play important roles in tumor angiogenesis including breast cancer. A series of 2,3-diaryl isoquinolinone derivatives were designed and synthesized targeting both estrogen receptor α (ERα) and VEGFR-2. Bioactivity evaluation showed that compounds 7c, 7d and 7f exhibited significant anti-proliferative and anti-angiogenesis activities via ERα and VEGFR-2 dependent mechanisms.

Prediction model and assessment of malnutrition in patients with stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) combined with malnutrition results in decreased exercise capacity and a worse quality of life. We aimed to develop an observational case-control study to explore the effective and convenient method to identify potential individuals is lacking. This study included data from 251 patients with COPD and 85 participants in the control group. Parameters and body composition were compared between groups, and among patients with varied severity. The LASSO approach was employed to select the features for fitting a logistic model to predict the risk of malnutrition in patients with stable COPD. Patients with COPD exhibited significantly lower 6-min walk distance (6MWD), handgrip strength, fat-free mass index (FFMI), skeletal muscle mass (SMM) and protein. The significant predictors identified following LASSO selection included 6MWD, waist-to-hip ratio (WHR), GOLD grades, the COPD Assessment Test (CAT) score, and the prevalence of acute exacerbations. The risk score model yielded good accuracy (C-index, 0.866 [95% CI 0.824-0.909]) and calibration (Brier score = 0.150). After internal validation, the adjusted C-index and Brier score were 0.849, and 0.165, respectively. This model may provide primary physicians with a simple scoring system to identify malnourished patients with COPD and develop appropriate rehabilitation interventions.

Xanthohumol and echinocystic acid induces PSTVd tolerance in tomato.

Tomato is a popular vegetable worldwide; its production is highly threatened by infection with the potato spindle tuber viroid (PSTVd). We obtained the full-length genome sequence of previously conserved PSTVd and inoculated it on four genotypes of semi-cultivated tomatoes selected from a local tomato germplasm resource. SC-5, which is a PSTVd-resistant genotype, and SC-96, which is a PSTVd-sensitive genotype, were identified by detecting the fruit yield, plant growth, biomass accumulation, physiological indices, and PSTVd genome titer after PSTVd inoculation. A non-target metabolomics study was conducted on PSTVd-infected and control SC-5 to identify potential anti-PSTVd metabolites. The platform of liquid chromatography-mass spectrometry detected 158 or 123 differential regulated metabolites in modes of positive ion or negative ion. Principal component analysis revealed a clear separation of the global metabolite profile between PSTVd-infected leaves and control regardless of the detection mode. The potential anti-PSTVd compounds, xanthohumol, oxalicine B, indole-3-carbinol, and rosmarinic acid were significantly upregulated in positive ion mode, whereas echinocystic acid, chlorogenic acid, and 5-acetylsalicylic acid were upregulated in negative ion mode. Xanthohumol and echinocystic acid were detected as the most upregulated metabolites and were exogenously applied on PSTVd-diseased SC-96 seedlings. Both xanthohumol and echinocystic acid had instant and long-term inhibition effect on PSTVd titer. The highest reduction of disease symptom was induced by 2.6 mg/L of xanthohumol and 2.0 mg/L of echinocystic acid after 10 days of leaf spraying, respectively. A superior effect was seen on echinocystic acid than on xanthohumol. Our study provides a statistical basis for breeding anti-viroid tomato genotypes and creating plant-originating chemical preparations to prevent viroid disease.

Structural Evaluations of a Selective Human STINGA230 Agonist and Its Use in Macrophage Immunotherapies.

Previously we identified a non-nucleotide agonist BDW568 that selectively activates the human STINGA230 allele. Here, we further characterized the mechanism of BDW568 and highlighted its potential use for selectively controlling the activation of engineered macrophages that constitutively express STINGA230 as a genetic adjuvant. We obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite) at 1.95 Å resolution. Structure-activity relationship studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining activity. We demonstrated that BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230. In contrast, BDW568 could not stimulate innate immune responses in human primary peripheral blood mononuclear cells in healthy donors in the absence of a STINGA230 allele. This high STING variant specificity suggested a promising application of STINGA230 agonists in macrophage-based therapeutic approaches.

Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5' untranslated region.

One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5' UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2'-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5' UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5' UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.

The Mechanism of Dendrite Formation in a Solid-State Transformation of High Aluminum Fe-Al Alloys.

The mechanism of solid-state dendrite formation in high-aluminum Fe-Al alloys is not clear. Applying an in-situ observation technique, the real-time formation and growth of FeAl solid-state dendrites during the eutectoid decomposition of the high-temperature phase Fe5Al8 is visualized. In-situ experiments by HT-CSLM reveal that proeutectoid FeAl usually does not preferentially nucleate at grain boundaries regardless of rapid or slow cooling conditions. The critical radii for generating morphological instability are 1.2 µm and 0.9 µm for slow and rapid cooling, respectively. The morphology after both slow and rapid cooling exhibits dendrites, while there are differences in the size and critical instability radius Rc, which are attributed to the different supersaturation S and the number of protrusions l. The combination of crystallographic and thermodynamic analysis indicates that solid-state dendrites only exist on the hypoeutectoid side in high-aluminum Fe-Al alloys. A large number of lattice defects in the parent phase provides an additional driving force for nucleation, leading to coherent nucleation from the interior of the parent phase grains based on the orientation relationship {3¯30}Fe5Al8//{1¯10}FeAl, <111¯>Fe5Al8//<111¯>FeAl. The maximum release of misfit strain energy leads to the preferential growth of the primary arm of the nucleus along <111¯> {1¯10}. During the rapid cooling process, a large supersaturation is induced in the matrix, driving the Al atoms to undergo unstable uphill diffusion and causing variations in the concentration gradient as well as generating constitutional undercooling, ultimately leading to morphological instability and the growth of secondary arms.

Structural and Biological Evaluations of a Non-Nucleoside STING Agonist Specific for Human STINGA230 Variants.

Previously we identified a non-nucleotide tricyclic agonist BDW568 that activates human STING (stimulator of interferon genes) gene variant containing A230 in a human monocyte cell line (THP-1). STINGA230 alleles, including HAQ and AQ, are less common STING variants in human population. To further characterize the mechanism of BDW568, we obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite of BDW568) at 1.95 Å resolution and found the planar tricyclic structure in BDW-OH dimerizes in the STING binding pocket and mimics the two nucleobases of the endogenous STING ligand 2',3'-cGAMP. This binding mode also resembles a known synthetic ligand of human STING, MSA-2, but not another tricyclic mouse STING agonist DMXAA. Structure-activity-relationship (SAR) studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining the compound's activity. BDW568 could robustly activate the STING pathway in human primary peripheral blood mononuclear cells (PBMCs) with STINGA230 genotype from healthy individuals. We also observed BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230, suggesting its potential use to selectively activate genetically engineered macrophages in macrophage-based approaches, such as chimeric antigen receptor (CAR)-macrophage immunotherapies.

Diagnostic and prognostic performance of plasma neurofilament light chain in multiple system atrophy: a cross-sectional and longitudinal study.

BACKGROUND: The longitudinal dynamics of neurofilament light chain (NfL) in multiple system atrophy (MSA) were incompletely illuminated. This study aimed to explore whether the plasma NfL (pNfL) could serve as a potential biomarker of clinical diagnosis and disease progression for MSA. METHODS: We quantified pNfL concentrations in both a large cross-sectional cohort with 214 MSA individuals, 65 PD individuals, and 211 healthy controls (HC), and a longitudinal cohort of 84 MSA patients. Propensity score matching (PSM) was used to balance the age between the three groups. The pNfL levels between groups were compared using Kruskal-Wallis test. Linear mixed models were performed to explore the disease progression-associated factors in longitudinal MSA cohort. Random forest model as a complement to linear models was employed to quantify the importance of predictors. RESULTS: Before and after matching the age by PSM, the pNfL levels could reliably differentiate MSA from HC and PD groups, but only had mild potential to distinguish PD from HC. By combining linear and nonlinear models, we demonstrated that pNfL levels at baseline, rather than the change rate of pNfL, displayed potential prognostic value for progression of MSA. The combination of baseline pNfL levels and other modifiers, such as subtypes, Hoehn-Yahr stage at baseline, was first shown to improve the diagnosis accuracy. CONCLUSIONS: Our study contributed to a better understanding of longitudinal dynamics of pNfL in MSA, and validated the values of pNfL as a non-invasive sensitive biomarker for the diagnosis and progression. The combination of pNfL and other factors is recommended for better monitoring and prediction of MSA progression.

Corrigendum: Inhibition of SARS-CoV-2 by Targeting Conserved Viral RNA Structures and Sequences.

[This corrects the article DOI: 10.3389/fchem.2021.802766.].

Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform.

Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways.

Coffin-Siris syndrome in two chinese patients with novel pathogenic variants of ARID1A and SMARCA4.

BACKGROUND: Coffin-Siris syndrome (CSS) is a rare congenital syndrome characterized by developmental delay, intellectual disability, microcephaly, coarse face and hypoplastic nail of the fifth digits. Heterozygous variants of different BAF complex-related genes were reported to cause CSS, including ARID1A and SMARCA4. So far, no CSS patients with ARID1A and SMARCA4 variants have been reported in China. OBJECTIVE: The aim of the current study was to identify the causes of two Chinese patients with congenital growth deficiency and intellectual disability. METHODS: Genomic DNA was extracted from the peripheral venous blood of patients and their family members. Genetic analysis included whole-exome and Sanger sequencing. Pathogenicity assessments of variants were performed according to the guideline of the American College of Medical Genetics and Genomics. The phenotypic characteristics of all CSS subtypes were summarized through literature review. RESULTS: We identified two Chinese CSS patients carrying novel variants of ARID1A and SMARCA4 respectively. The cases presented most core symptoms of CSS except for the digits involvement. Additionally, we performed a review of the phenotypic characteristics in CSS, highlighting phenotypic varieties and related potential causes. CONCLUSIONS: We reported the first Chinese CSS2 and CSS4 patients with novel variants of ARID1A and SMARCA4. Our study expanded the genetic and phenotypic spectrum of CSS, providing a comprehensive overview of genotype-phenotype correlations of CSS.