HEK293T Cells Research Articles

Isoxazole-Carboxamide Modulators of GluA2-Containing AMPA Receptors in Parkinson's Disease.

AMPA receptors, especially GluA2-containing subunits, play a pivotal role in minimizing calcium permeability and excitotoxicity as part of the critical developments in the pathogenesis of Parkinson's disease (PD). AMPA receptor modulation has been considered one promising therapeutic avenue to counteract glutamate neurotoxicity. The present study explored the potential of fluorophenyl-isoxazole-carboxamide (ISX) derivatives, including ISX-1 and its analogs, as modulators of GluA2-containing AMPA receptor activity. These were synthesized and then characterized to be used to effectively modulate AMPA receptor kinetics using advanced electrophysiological techniques in HEK293T cells. ISX-11 exhibited the most potent inhibitory effect among the tested compounds, reducing GluA2 and GluA2/3 currents (p < 0.001), with IC50 values of 4.4 µM and 4.62 µM, respectively. ISX-8 also showed significant inhibition (p < 0.001), with IC50 values of 4.6 µM and 4.79 µM. Additionally, ISX-11 and ISX-8 significantly increased deactivation rates by 2.5- and 2-fold, respectively, while decreasing desensitization rates by similar magnitudes. Fluorophenyl, methoxy, and tert-butyl substituents have been most important in achieving excellent pharmacodynamic and pharmacokinetic properties for AMPA modulators. This report supplements earlier reports on how the structure can affect the activity of various isoxazole derivatives, displaying reduced excitotoxic injury to nigrostriatal dopaminergic neurons relevant to PD.

Global analysis by LC-MS/MS of N6-methyladenosine and inosine in mRNA reveal complex incidence.

The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.

Elucidating interplay between myrcene and cannabinoid receptor 1 receptors to produce antinociception in mouse models of neuropathic pain.

The need for nonaddictive and effective treatments for chronic pain are at an all-time high. Historical precedence, and now clinical evidence, supports the use of cannabis for alleviating chronic pain. A plethora of research on delta-9-tetrahydrocannabinol exists, yet cannabis is comprised of a multitude of constituents, some of which possess analgesic potential, that have not been systematically investigated, including the terpene myrcene. Myrcene attenuates pain hypersensitivity in preclinical models and is one of the most abundant terpenes found in cannabis. Despite these findings, it remains unclear how myrcene elicits these effects on nociceptive systems. The present study uses a male and female mouse model of neuropathic pain as well as in vitro experiments with HEK293T cells to explore these questions. We first demonstrate myrcene (1-200 mg/kg i.p.) dose-dependently increases mechanical nociceptive thresholds, where potency was greater in female compared with male pain mice. Testing canonical tetrad outcomes, mice were tested for hypolocomotion and hypothermia after myrcene administration. Myrcene did not alter locomotion or temperature, but female pain mice showed a conditioned place aversion to myrcene. A cannabinoid receptor 1 (CB1) antagonist inhibited myrcene's anti-allodynia. By contrast, in vitro cell culture experiments using a TRUPATH assay revealed myrcene does not directly activate CB1 receptors nor alter CB1 receptor activity elicited by CB1 agonist (CP 55,940) or endocannabinoids (anandamide or 2-arachidonoylglycerol). Understanding engagement of CB1 receptors in pain modulation and myrcene's mechanism of action warrants further study to understand the diversity of cannabis pharmacology and to further the frontier of pain research.

Novel biallelic TK2 mutations cause mitochondrial DNA depletion syndrome with infantile early-onset lipid storage myopathy

BackgroundMutations in the TK2 gene are strongly associated with mitochondrial DNA depletion syndrome (MDS), a severe condition with high mortality and poor outcomes. Although many MDS cases are reported, those linked to TK2 mutations with lipid deposition are rare. Large deletions in the TK2 gene are even rarer.MethodsWe conducted whole-exome sequencing to find the gene linked to MDS, followed by genomic and structural analyses, histopathological, and functional analyses to assess the mutations' pathogenicity. Additionally, a HEK293T cell model with TK2 mutations was created to investigate the impact of large deletions on mitochondrial function.ResultsThe patient was found to have a novel compound heterozygous mutation in the TK2 gene, consisting of a large deletion spanning exons 5–10 (E5-E10 del) and a previously reported missense mutation (c.311C > A, p.Arg104His). Analysis of the patient's muscle tissue demonstrated a marked reduction in mtDNA content and a significant impairment in overall mitochondrial function. In the HEK293T cell model, the group with the deletion mutation exhibited a notable reduction in TK2 protein expression and levels of mitochondrial complex subunits when compared to the control group. Furthermore, there was an observed increase in ROS levels, a decrease in ATP production, and compromised mitochondrial respiratory chain function. Moreover, we conducted a comprehensive review of the previously reported genotypic and phenotypic spectrum of TK2 mutations in the literature.ConclusionsThis case report underscores the detrimental impact of large fragment deletion mutations in the TK2 gene and elucidates their role in the pathogenesis of MDS. It broadens the spectrum of known TK2 mutations and enhances our understanding of the structural and functional consequences of these mutations.

A competitive ELISA based on nanobodies for the detection of serum neutralizing antibodies against porcine epidemic diarrhea virus

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), can induce 80–100% mortality in newborn piglets; therefore, specific and rapid detection methods are important for the prevention of this viral infection. In particular, methods for detecting neutralizing antibodies (nAbs) can be used to evaluate the immunization effect of PEDV vaccines. The spike protein of PEDV (PEDV-S) has been universally used as an antigen to develop immunoassays to detect nAbs. Nanobodies (Nbs) offer advantages such as ease of genetic engineering and low production costs, making them promising for diagnostic applications. In this study, PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels. A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase (HRP) in HEK293T cells. A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay (cELISA) to detect anti-PEDV nAbs. Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration. The optimal dilution of PEDV-S-Nb13-HRP was 1:200, and the optimal serum dilution was 1:10. The cutoff value of cELISA was determined as 28.1%, demonstrating high specificity, repeatability, stability, and good agreement rates with two commercial ELISA kits (93.6%) and a serum neutralization test (96.34%). Additionally, the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit. These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.

In vitro characterization of polymeric nanoparticles encapsulating plasmid DNA for gene therapy

Gene therapy is a promising clinical approach for treating or preventing genetic diseases by directly targeting disease-causing mutations. Despite the potential for gene therapy in addressing a broad range of genetic diseases, the development of these techniques remains in its early stages. A key challenge in the field is the development of approaches that efficiently deliver modified genetic material to intended biological targets, but minimize degradation and off-target effects. Biomedical polymeric nanoparticles (PNPs) can enhance gene therapy delivery by encapsulating, protecting, and releasing therapeutic compounds within target cells. However, their clinical translation is hindered by poor efficacy and storage instability. To address these challenges, we formulated Envoyer nanoparticles, a novel natural polymer-based delivery system. Here, we evaluate the stability and transfection efficiency of Envoyer nanoparticles in multiple human cell lines. In HEK293T cells, Envoyer nanoparticles (50–70 nm) encapsulating green fluorescent protein (GFP) plasmid DNA (pDNA) achieved a transfection efficiency of >80%, outperforming lipofectamine controls (∼60%). In PANC1 pancreatic cancer cells, known for being difficult to transfect, Envoyer nanoparticles also demonstrated superior efficacy compared to lipofectamine controls. Notably, commonly used commercially available nanoparticles failed to produce GFP expression under similar conditions. Preliminary mechanistic studies suggest that Envoyer nanoparticle internalization occurs via clathrin-independent endocytosis. Furthermore, we show that Envoyer nanoparticles maintain stability and transfection efficiency after storage for 2 months at 4 °C and −80 °C. Collectively, these findings highlight the potential of Envoyer nanoparticles as a stable and efficient gene delivery platform for future clinical applications in gene therapy.

Functional Analysis of Mature Activin A Produced by Enterokinase in Plant Cells

Molecular farming for producing biopharmaceuticals in plants is considered an excellent method to replace some of the production methods currently used, and a significant number of recombinant proteins have already shown the potential to facilitate this. In particular, production of activin A, which has a variety of important biological functions in humans, is limited. The purpose of this study was to develop a safe, stable, and efficient plant-based in vitro production system for activin A, assess its biological activity in cancer cells, and demonstrate its potential for use in cancer research. We evaluated the expression and production of activin A in plant cells through a mass culture and secretion system. The formation of mature activin A homodimers, produced by enterokinase, was also assessed. Southern blot and inverse PCR were performed to investigate the gene insertion sites in the plants, and the stability of activin A was evaluated over six months under various pH conditions. The activity of plant-derived activin A was analyzed in HEK293T, Huh7, MCF7, and MDA-MB-231 cancer cell lines using luciferase reporter, migration, phosphorylation, and gelatin zymography assays. We developed cell line #71, which showed the highest levels of mature activin A expression (8.44 μg/g calli fresh weight) and had multicopy gene insertions. Pro-activin A was converted to mature activin A using enterokinase. We demonstrated that the optimal stability of plant-derived activin A was maintained for six months at pH 7 below 4 °C. Plant-derived activin A significantly enhanced activin A signaling activity in HEK293T, Huh7, and MCF7 cancer cells. Additionally, we confirmed that plant-derived activin A inhibited the growth of Huh7 cancer cells by activating the Smad pathway without affecting the MAPK pathway. Contrastingly, in MDA-MB-231 breast cancer cells, plant-derived activin A promoted cell migration. Our results confirm that plant-derived activin A, produced using a mass production system, exhibits full biological activity and affects cancer cell behavior in a manner similar to activin A derived from traditional mammalian systems. Furthermore, this study highlights the importance of considering cellular context when determining the functional outcomes of activin A treatment.

Minigene assay for verifying the splicing effects of the rare variants in the SLC12A2 gene at c.2930-1G>a

Background Hearing loss (HL) is a prevalent sensory impairment with a genetic basis. The SLC12A2 gene, encoding NKCC1, is vital for inner ear ion balance. The c.2930-1G > A variant is a novel mutation potentially linked to sensorineural hearing loss. Objectives To investigate the splicing and protein expression effects of the c.2930-1G>A variant in SLC12A2 and its role in hearing loss. Material and methods Minigene assays and plasmid transfection in HEK-293T and Hela cells were used to study splicing. Protein expression and modification were also assessed. Results The c.2930-1G>A variant caused partial exon skipping, altering mRNA splicing in both cell types. This suggests a potential involvement in sensorineural hearing loss. Protein analysis showed the E21del mutation increased expression without altering modification patterns, whereas the 2930_2977del mutation reduced both, possibly impacting stability or modification sites. Conclusions and significance The c.2930-1G>A variant likely contribute to hearing loss by disrupting splicing and protein expression. Currently a Variant of Uncertain Significance (VUS), its pathogenicity is supported by these findings. Further research is needed to confirm its role, emphasizing the need for integrated genetic and clinical data in auditory disorders management.

Receptor tyrosine kinases CAD96CA and FGFR1 function as the cell membrane receptors of insect juvenile hormone.

Juvenile hormone (JH) is important to maintain insect larval status; however, its cell membrane receptor has not been identified. Using the lepidopteran insect Helicoverpa armigera (cotton bollworm), a serious agricultural pest, as a model, we determined that receptor tyrosine kinases (RTKs) cadherin 96ca (CAD96CA) and fibroblast growth factor receptor homologue (FGFR1) function as JH cell membrane receptors by their roles in JH-regulated gene expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of JH intracellular receptor MET1 and cofactor Taiman, and high affinity to JH III. Gene knockout of Cad96ca and Fgfr1 by CRISPR/Cas9 in embryo and knockdown in various insect cells, and overexpression of CAD96CA and FGFR1 in mammalian HEK-293T cells all supported CAD96CA and FGFR1 transmitting JH signal as JH cell membrane receptors.

Effect of N1-methyladenosine in the quantification of RNA.

The reverse transcription (RT) of RNA to cDNA is a key step for the quantification of nucleic acid molecules in numerous basic research and medical diagnosis. Although multiple sources of errors have been considered, little is known about the impact of RNA modifications on the validity of genes of interest for quantitative RT-PCR. Here, we evaluated the influence of RNA modifications of N1-methyladenosine (m1A) on the validity of the RT step by quantifying two RNAs with commercial reverse transcriptase and RNA sample from HEK-293T cells or in vitro transcription. Our findings prove that RNA modification of m1A is a source of RT variability as it acts as an arrest signal of RT at its position, in turn affecting the corresponding RNA quantification.

Transient receptor potential vanilloid-1 (TRPV1) is a mediator of noise-induced neural damage in zebrafish and mice.

Sound pollution (noise) is an increasing environmental concern, particularly associated with neurological and neurobehavioral abnormalities. However, the molecular mechanisms underlying noise-induced neural damage remain unclear. In this study, we conducted transcriptional profiling of zebrafish to investigate the mechanisms underlying acoustic stimulation (1,000 Hz, 130 dB). RNA sequencing and subsequent experiments revealed that TRPV1 is an important mediator of noise-induced neural damage in HuC(elavl3)-GFP transgenic zebrafish. The results demonstrated that inhibiting TRPV1 significantly mitigated noise-induced neural damage in zebrafish with trpv1 gene RNAi and in mice with Trpv1 knockout (Trpv1-/-). Specifically, TRPV1 antagonism significantly reduced neural damage in zebrafish and mice under noise exposure. Furthermore, activated TRPV1 could induce endoplasmic reticulum stress, leading to apoptosis and resulting in neural damage in mice and HEK293T cells. The findings of this study not only enhance our understanding of the molecular mechanisms underlying sound-induced neural damage but also highlight a novel target for drug intervention.

A Hypothetical Protein Fragment from Large Yellow Croaker (Larimichthys crocea) Demonstrates Significant Activity Against Both Bacterial and Parasite

Antimicrobial peptides (AMPs) are biocompatible and biodegradable, making them an attractive alternative to traditional antimicrobial agents and chemical preservatives. Here, a novel α-helix amphiphilic anionic AMP Lc149 was screened from a large yellow croaker (Larimichthys crocea) using a Bacillus subtilis expression system. Lc149 is a hypothesized protein fragment not annotated in the genome of a large yellow croaker. Both extracellular protein and recombinant Lc149 (rLc149) exhibited significant killing effects against Gram-negative Escherichia coli and Vibrio harveyi. Scanning and transmission electron microscopy revealed that rLc149 had the ability to disrupt bacterial cell membranes, causing irregular cell morphology, severe cell membrane damage, cytoplasm agglutination, and intracellular content leakage. Confocal laser scanning microscopy and flow cytometry further confirmed bacterial cell destruction and mortality rates of over 80%. Gel retardation assays and SDS-PAGE electrophoresis showed that rLc149 was unable to bind to bacterial DNA, but did reduce bacterial protein contents. Additionally, rLc149 maintained antibacterial activity against E. coli and V. harveyi upon exposure to temperatures of 25–100 °C, UV radiation time of 0–60 min, pH levels of 3–12, and different proteases. Biosafety assays revealed low hemolytic toxicity to erythrocytes of large yellow croaker, rabbit, and shrimp, and low cytotoxicity to large yellow croaker kidney cells and HEK 293T cells. More deeply, rLc149 also possessed significant killing activity against parasites. Therefore, rLc149 can be considered an antibacterial and antiparasitic drug in fisheries.

SCN8A Epileptic Encephalopathy Mutation Displays a Loss-of-Function Phenotype and Distinct Insensitivity to Valproate.

Voltage-gated sodium channels are the main targets of antiepileptic drugs, such as sodium valproate (VPA). Single nucleotide polymorphisms (SNPs) in the Nav1.6 isoform (SCN8A) have been reported to be closely associated with motor dysfunction in pediatric akathisia epileptica. In this study, we conducted a genetic screening of pediatric patients with seizures treated solely with VPA and identified two novel missense mutations of SCN8A (A1534V and Q1853H). Electrophysiological results revealed that the peak currents of the A1534V variant were smaller compared to that of the wild-type (WT) channel. The A1534V variant also caused a positive shift in the I-V curve, indicating a change in the voltage dependence of activation compared to the WT channels. In contrast, VPA induced a significant negative shift in the inactivation of both WT and A1534V mutant. However, the inhibition of currents by VPA was weaker in the A1534V variant than in WT. Furthermore, the recovery time constant of the A1534V variant was shorter than that of WT when treated with VPA. Regrettably, although the Q1853H variant can be expressed in HEK293T cells, the detected current is too small (approximately 50 pA). In conclusion, our results suggest that the A1534V mutation is a novel loss-of-function variant that exhibits moderate insensitivity to VPA. These results underscore the importance of Nav1.6 as a key target in epilepsy and highlight the necessity of analyzing its role in the pathological process.

Low-temperature enhances production of severe fever with thrombocytopenia syndrome virus virus-like particles

Tick-borne severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease caused by the SFTS virus (SFTSV). Serological assays based on the nucleocapsid protein and partial glycoprotein of this virus have been used for detecting SFTSV infections in humans and animals. However, whether the complete SFTSV glycoprotein (Gn/Gc) can induce the assembly of virus-like particles (VLPs) which can be used for serological surveillance and vaccine production remains unclear. In this study, we successfully expressed and secreted SFTSV Gn/Gc antigens by using a single plasmid encoding the complete glycoprotein sequence of the dominant genotype B virus. HEK293T and COS-1 cells were transfected with the aforementioned plasmid; cultivating these cells at 32 °C, instead of 37 °C, led to 4.0- and 3.3-fold higher antigen recovery, respectively. The secreted Gn/Gc antigens at 32 °C retained epitopes resembling those of the virion; these epitopes were recognized by a SFTS human–derived monoclonal antibody. Sucrose density gradient centrifugation, followed by transmission electron microscopy, confirmed the formation of VLPs with a diameter of approximately 100 nm. Overall, our findings highlight the potential of SFTSV VLPs for serological surveillance and vaccine development.Key points• Cultivating transfected cells at 32 °C boosts SFTSV glycoprotein production.• Complete SFTSV glycoprotein expression facilitates virus-like particle assembly.• The assembly does not require any other viral proteins or RNA.

The Kv2.2 channel mediates the inhibition of prostaglandin E2 on glucose-stimulated insulin secretion in pancreatic β-cells

Prostaglandin E2 (PGE2) is an endogenous inhibitor of glucose-stimulated insulin secretion (GSIS) and plays an important role in pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). This study aimed to explore the underlying mechanism by which PGE2 inhibits GSIS. Our results showed that PGE2 inhibited Kv2.2 channels via increasing PKA activity in HEK293T cells overexpressed with Kv2.2 channels. Point mutation analysis demonstrated that S448 residue was responsible for the PKA-dependent modulation of Kv2.2. Furthermore, the inhibitory effect of PGE2 on Kv2.2 was blocked by EP2/4 receptor antagonists, while mimicked by EP2/4 receptor agonists. The immune fluorescence results showed that EP1–4 receptors are expressed in both mouse and human β-cells. In INS-1(832/13) β-cells, PGE2 inhibited voltage-gated potassium currents and electrical activity through EP2/4 receptors and Kv2.2 channels. Knockdown of Kcnb2 reduced the action potential firing frequency and alleviated the inhibition of PGE2 on GSIS in INS-1(832/13) β-cells. PGE2 impaired glucose tolerance in wild-type mice but did not alter glucose tolerance in Kcnb2 knockout mice. Knockout of Kcnb2 reduced electrical activity, GSIS and abrogated the inhibition of PGE2 on GSIS in mouse islets. In conclusion, we have demonstrated that PGE2 inhibits GSIS in pancreatic β-cells through the EP2/4-Kv2.2 signaling pathway. The findings highlight the significant role of Kv2.2 channels in the regulation of β-cell repetitive firing and insulin secretion, and contribute to the understanding of the molecular basis of β-cell dysfunction in diabetes.

The Kv2.2 channel mediates the inhibition of prostaglandin E2 on glucose-stimulated insulin secretion in pancreatic β-cells.

Prostaglandin E2 (PGE2) is an endogenous inhibitor of glucose-stimulated insulin secretion (GSIS) and plays an important role in pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). This study aimed to explore the underlying mechanism by which PGE2 inhibits GSIS. Our results showed that PGE2 inhibited Kv2.2 channels via increasing PKA activity in HEK293T cells overexpressed with Kv2.2 channels. Point mutation analysis demonstrated that S448 residue was responsible for the PKA-dependent modulation of Kv2.2. Furthermore, the inhibitory effect of PGE2 on Kv2.2 was blocked by EP2/4 receptor antagonists, while mimicked by EP2/4 receptor agonists. The immune fluorescence results showed that EP1-4 receptors are expressed in both mouse and human β-cells. In INS-1(832/13) β-cells, PGE2 inhibited voltage-gated potassium currents and electrical activity through EP2/4 receptors and Kv2.2 channels. Knockdown of Kcnb2 reduced the action potential firing frequency and alleviated the inhibition of PGE2 on GSIS in INS-1(832/13) β-cells. PGE2 impaired glucose tolerance in wild-type mice but did not alter glucose tolerance in Kcnb2 knockout mice. Knockout of Kcnb2 reduced electrical activity, GSIS and abrogated the inhibition of PGE2 on GSIS in mouse islets. In conclusion, we have demonstrated that PGE2 inhibits GSIS in pancreatic β-cells through the EP2/4-Kv2.2 signaling pathway. The findings highlight the significant role of Kv2.2 channels in the regulation of β-cell repetitive firing and insulin secretion, and contribute to the understanding of the molecular basis of β-cell dysfunction in diabetes.

Pharmacological characterization of cannabidiol as a negative allosteric modulator of the 5-HT2A receptor.

Pharmacological characterization of cannabidiol as a negative allosteric modulator of the 5-HT2A receptor.

Thallium induces metallothionein gene expression in Huh-7 human hepatoma cells.

Thallium induces metallothionein gene expression in Huh-7 human hepatoma cells.

ChNLRC4, a cytoplasmic pattern recognition receptor, activates the pyroptosis signaling pathway in Mollusca.

ChNLRC4, a cytoplasmic pattern recognition receptor, activates the pyroptosis signaling pathway in Mollusca.

Two Nonsense GLI3 Variants Are Identified in Two Chinese Families With Polydactyly.

Polydactyly is a prevalent limb deformity with an autosomal dominant inheritance pattern, manifesting in both syndromic and nonsyndromic forms. It exhibits significant etiological and clinical diversity. This study aims to identify the pathogenic cause in two patients with sub-PHS (sub-Pallister-Hall Syndrome) and PAP (postaxial polydactyly), respectively, from two Chinese pedigrees. Exome sequencing was performed on patients to screen for potential pathogenic variants. Subsequently, these variants were validated by Sanger sequencing. The c.3342dupC and c.4431dupT mutant plasmids were transfected into HEK293T cells, and the effects of GLI3 mutations on transcription and protein levels were analyzed via qRT-PCR and Western blot. Additionally, Swiss Model was utilized to predict the effects of mutations on protein tertiary structure. The mutations of GLI3 (NM_000168.6: c.3342dupC; p. A1115Rfs*14) (NM_000168.6: c.4431dupT; p. Glu1478Ter) were identified in affected individuals. These mutations were present exclusively in the patients and absent in the healthy individuals. No significant difference in transcription levels between the mutations and wild type was observed. Functional analysis revealed that the truncated variants p. A1115Rfs*14 and p. Glu1478Ter exhibited reduced molecular weight and potential functional impairment due to protein retention. The mutations p. A1115Rfs*14 and p. Glu1478Ter in GLI3 may account for sub-PHS and PAP in the two patients, respectively. This finding expands the mutation and phenotype spectrum associated with GLI3, providing valuable insights for the clinical diagnosis of polysyndactyly.