OBJECTIVE: To analyze the clinical phenotype and pathogenic mechanism of the ARSL gene variant in a fetus with nasal bone aplasia. METHODS: A 34-year-old pregnant woman who attended Quzhou Maternal and Child Health Care Hospital on January 3, 2023 was selected as the study subject. Whole exome sequencing (WES) was performed on the fetus. Bioinformatics analysis was carried out to identify and prioritize candidate gene variants, followed by Sanger sequencing for familial validation. A mutant plasmid expression vector was constructed and subsequently transfected into HEK293T cells to preliminarily investigate the pathogenetic mechanism of the identified variant. Additionally, a comprehensive review of literature was conducted to systematically summarize the associated clinical phenotypes. This study was approved by the Medical Ethics Committee of Quzhou Maternal and Child Health Care Hospital (Ethics No.: KY-2023-11). RESULTS: WES revealed that the fetus harbored a c.827del (p.L276Rfs*48) variant of the ARSL gene, for which its mother was heterozygous. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic(PVS1+PM2_Supporting). In vitro cellular function studies demonstrated that this variant can result in a substantial decrease in the expression of mutant mRNA, thereby preventing the production of normal ARSL protein. Clinical phenotypes resulting from ARSL gene variants exhibited considerable diversity, with nasal hypoplasia being the most common manifestation. CONCLUSION The c.827del (p.L276Rfs*48) variant of the ARSL gene can lead to degradation of mRNA via the nonsense-mediated mRNA decay pathway, resulting in reduced levels of ARSL protein. The pathogenetic mechanism underlying the ARSL gene variant may be associated with its haploinsufficiency effect.
Humans ; Female ; Pregnancy ; Adult ; Frameshift Mutation ; HEK293 Cells ; Nasal Bone/abnormalities* ; Fetus/abnormalities* ; Exome Sequencing

OBJECTIVE: To analyze the functional impact of a rare heterozygous variant of SOS1 gene (c.283G>A, p.E95K) identified in a fetus with cervical cystic hygroma and to explore its association with the disease phenotype. METHODS: A pedigree analysis was carried out to evaluate the co-segregation of the variant with the disease phenotype. Bioinformatic tools were employed to assess the conservation, protein structure and stability. Functional validation was conducted on HEK293T cells using fluorescence quantitative reverse transcription-PCR and Western blotting to measure the expression of SOS1 and phosphorylation levels of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase. A literature review of previously reported disease-associated SOS1 variants was also carried out. This study has been approved by the Medical Ethics Committee of West China Second University Hospital, Sichuan University (Ethics No.: 201940). RESULTS: The variant was inherited from the husband of the woman with distinctive facial features and has co-segregated with the phenotype. Bioinformatics analysis indicated that the variant is located in a highly conserved region, and that p.E95K could disrupt key amino acid interactions and protein stability. Multiple bioinformatic predictions consistently suggested the pathogenicity of this variant. Functional assays demonstrated reduced SOS1 protein expression and decreased ERK phosphorylation. CONCLUSION This study has revealed the functional impact of the SOS1 c.283G>A (p.E95K) variant, suggesting that it may contribute to the developmental phenotypes through a haploinsufficiency mechanism.
Humans ; SOS1 Protein/chemistry* ; Female ; HEK293 Cells ; Male ; Pedigree ; Phenotype ; Adult

OBJECTIVE

To evaluate cone- and rod-specific nonperfusion indices (CPI, RPI) on ultrawide field fluorescein angiography (UWF-FA) and their correlation with diabetic retinopathy (DR) severity and center-involving diabetic macular edema (ciDME).

Sixty-nine eyes of 43 patients with diabetes underwent UWF color photography (UWF-CP), UWF-FA, and macular optical coherence tomography (OCT). DR severity was graded by a masked reader on UWF-CP. UWFFA images were segmented into posterior pole (≤10 mm from fovea), mid-periphery (10–15 mm) and far periphery (>15 mm), and into the macula, within ETDRS fields and extended peripheral fields. Ischemic areas were quantified using ImageJ to calculate nonperfusion index (NPI), CPI, and RPI. ciDME was determined on OCT. Correlations were analyzed using Pearson coefficients (r).

DR severity correlated significantly with global NPI (r = 0.56, p < 0.0001) and across all retinal zones (r = 0.35– 0.57). Both CPI (global: r = 0.56, p < 0.0001; zones: r = 0.42–0.59) and RPI (global: r = 0.55, p < 0.0001; zones: r = 0.40–0.59) showed similar associations. ciDME presence was also correlated with NPI (global: r = 0.40, p = 0.0014; zones: r = 0.42–0.54), CPI (global: r = 0.47, p = 0.0001; zones: r = 0.32–0.55), and RPI (global: r = 0.46, p = 0.0002; zones: r = 0.33–0.54).

Cone- and rod-related retinal nonperfusion strongly correlate with DR severity and ciDME. UWF-FA may help identify high-risk eyes and guide surveillance of ischemia-related progression. Further studies are needed to define ischemic thresholds predictive of vision-threatening complications.

Introduction: Leydig cells (LCs) are the principal source of testosterone in males, underpinning male reproductive health and androgendependent physiology. Precise isolation of viable human LCs is essential for mechanistic steroidogenesis research and cell-based therapeutic development. Existing protocols rely on density-gradient centrifugation, which is technically demanding and often compromises yield and viability. Here, we describe a simplified density-gradient-free approach using differential adhesion to enrich functional human LCs from testicular tissue. Methodology: Human testicular fragments (~5 mm³) were minced and enzymatically digested with collagenase IV (2 mg/mL) at 37°C for 20 min under gentle agitation (100 RPM). The suspension was filtered (45 µm) and plated onto poly-L-lysine-coated T25 flasks. After 24 hours, non-adherent cells were removed by PBS washing. Viability exceeded 90% by trypan blue exclusion. Cells were maintained in DMEM/F12 with 10% FBS, 1% Antibiotic-Antimycotic, and 10 ng/mL luteinizing hormone (LH) to preserve the mature LC phenotype. Characterization employed immunofluorescence and flow cytometry using antibodies against SF-1, StAR, LHCGR, PDGFRA, and TEM-1. Testosterone secretion was quantified by ELISA under basal conditions and LH-stimulated conditions (10 ng/mL). Results: The protocol yielded ~1.25 × 10⁶ LCs per gram of tissue with >80% purity. Adherent HLCs displayed dense cytoplasmic lipid granules and intercellular networks consistent with active steroidogenesis. Flow cytometry confirmed 84.7% StAR⁺ cells, indicative of a robust steroidogenic population. A distinct progenitor subpopulation (PDGFRα⁺/TEM-1⁺) comprising ~28% of primary cultures, suggests retention of regenerative capacity. Basal testosterone secretion averaged 8.88 ng/mL per 24 hours, confirming preserved functional activity post-isolation. Conclusion This differential-adhesion protocol efficiently isolates functional human LCs without density-gradient media. Multimodal validation integrating immunofluorescence, flow cytometry, and ELISA confirms both phenotypic identity and steroidogenic competence, providing a reproducible and accessible platform for LC research and translational applications in male hypogonadism and androgen replacement.
Leydig Cells

Introduction: Hurthle cell adenoma is a rare benign thyroid neoplasm that can only be diagnosed through histopathological examination. Hurthle cell neoplasm typically presents as nonfunctioning cold nodule on thyroid scintigraphy. We report a rare case of Hurthle cell adenoma presenting with thyroid storm, with unusual findings of “cold” within “hot” thyroid nodule on scintigraphy. Case: A 73-year-old male with hypertension, chronic kidney disease, coronary artery disease, and Parkinson’s disease presented to the emergency department with fever and diarrhea. His temperature was 38.4°C, heart rate 106 bpm, and blood pressure 138/75 mmHg, with atrial fibrillation and signs of heart failure. The Burch-Wartofsky score was 50, consistent with thyroid storm. Laboratory tests revealed free thyroxine 4 37.8 pmol/L (NR 11.5–22.7), free thyroxine 3 5.6 pmol/L (NR 3.5–6.5), and thyroid-stimulating hormone <0.01 mIU/L (NR 0.55– 4.78). Thyroid autoantibodies, including anti-thyroid peroxidase, anti-thyroglobulin, and thyroid-stimulating immunoglobulins, were negative (<0.10 IU/L). The thyroid storm was precipitated by invasive Klebsiella syndrome with endophthalmitis and lung and liver abscess. He was treated with Lugol’s iodine, corticosteroid, antibiotics, and carbimazole. Ultrasound thyroid revealed a mixed cystic-solid nodule in the left thyroid lobe, measuring 2.3 × 3.3 × 4.3 cm (TIRADS category 3). Technetium-99m thyroid scintigraphy demonstrated a hyperfunctioning left thyroid nodule with a focal intranodular cold spot measuring 5.0 × 3.7 cm. Fine needle aspiration cytology of the nodule was benign follicular cells. Following stabilization with anti-thyroid treatment, he underwent left hemithyroidectomy. Histopathology examination revealed a Hurthle cell adenoma without capsular or vascular invasion. Postoperatively, he remained clinically euthyroid. Surveillance ultrasound performed 8 months later showed a normal right thyroid lobe, and lifelong surveillance was planned. Conclusion This case illustrates a rare and unusual presentation of thyroid storm caused by a toxic Hurthle cell adenoma containing an intranodular cold spot on scintigraphy. To our knowledge, only one similar case has been reported in the literature, and our case is the first to present with thyroid storm.
Oxyphil Cells ; Thyroid Crisis ; Adenoma

OBJECTIVE

The aim of this study was to qualitatively review the effects of genotoxicity and cytotoxicity on buccal mucosal epithelial cells after cone beam computed tomography (CBCT) exposure focusing on DNA damage and cell changes.

A literature search was carried out in PubMed, Wiley, Google Scholar, and Semantic Scholar for articles published in the last five years. In vivo studies that analyzed the DNA damage and cell changes on buccal mucosal epithelial cells, before and several days after CBCT exposure were included in this review. This review was prepared according to the PRISMA checklist for systematic review and the risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies tool.

A total of four studies were included in this review. The risk of bias analysis showed that all studies had generally good methodological quality. All the studies used buccal epithelial cells to analyze micronucleus (MN) as a parameter for DNA damage (genotoxicity), three of the studies also analyzed cytotoxicity using pyknotic nucleus and three studies analyzed karyolysis and karyorrhexis. All the studies consistently reported a significant increase in MN frequency, and cytotoxic effect were more evident before and 10-15 days after CBCT exposure.

This study demonstrated a significant impact on DNA and cell damage in oral mucosal cells following CBCT examination. The effect of ionizing radiation from CBCT has a more pronounced impact on cell damage than DNA damage.

BACKGROUND : Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that impacts several organ systems. Regulatory T cells (Tregs) are essential for maintaining immunological tolerance, and their dysregulation has been found linked to the pathogenesis of SLE. This study aimed to evaluate the correlation between CD4⁺CD25⁺FOXP3⁺ Treg levels and disease activity, as measured by the MEX-SLEDAI score, in patients with SLE. METHODS : A cross-sectional study was conducted involving 40 SLE patients treated in the Allergy and Immunology outpatient clinic and inpatient ward of Mohammad Hoesin Hospital from June 1, 2024 to August 31, 2024. Inclusion criteria were adults (≥18 years) diagnosed with SLE who provided informed consent. Exclusion criteria included other history autoimmune or immunocompromised conditions, pregnancy or lactation, hormonal contraceptive use, and active severe infection. CD4⁺CD25⁺FOXP3⁺ Treg cell levels were measured by flow cytometry. Disease activity was assessed using the MEX-SLEDAI score. Spearman’s correlation test was used to analyze the association between Treg levels and disease activity. RESULTS : Of 40 enrolled subjects (39 women, 1 man), 75% were aged 20–44 years. Mucocutaneous manifestations were the most common clinical feature (95%). Nephritis and hematologic involvement were most frequently observed in patients with severe disease activity. A strong negative correlation was identified between CD4⁺CD25⁺FOXP3⁺ Treg cell levels and MEX-SLEDAI scores (R = –0.72, p<0.001), indicating that lower Treg levels were associated with higher disease activity. CONCLUSIONS CD4⁺CD25⁺FOXP3⁺ Treg levels show a significant negative correlation with MEX-SLEDAI disease activity in SLE. Lower Treg cell levels reflect more severe disease activity.
disease progression ; flow cytometry ; systemic lupus erythematosus ; Treg cells

OBJECTIVE: To explore the pathogenic mechanism of a child with Waardenburg syndrome type 4C due to a c.661_664dup (p.P222Lfs*60) variant of SOX10 gene through in vitro experiments. METHODS: A child diagnosed at the Handan First Hospital was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples were collected from the child and his parents. Following extraction of genomic DNA, trio-whole exome sequencing was carried out. Pathogenicity of candidate variant was determined by bioinformatic analysis and reference to the guidelines from the American College of Medical Genetics and Genomics (ACMG). Candidate variant was verified by Sanger sequencing. Expression plasmids of wild-type SOX10 and the c.661_664dup (p.P222Lfs*60) variant were constructed and transiently transfected into 293T cells to determine the expression at the RNA and protein levels. The 293T cells transiently transfected with the wild-type/mutant SOX10 were treated with 10 ug/mL cycloheximide (CHX) for 0, 4, 8, 24 h, respectively, and the degradation rate of target protein was detected by Western blotting assay. This study has been approved by the Ethics Committee of Handan First Hospital (Ethics No. HDYY-LW-25053). RESULTS: The child was found to harbor a heterozygous c.661_664dup (p.P222Lfs*60) variant of the SOX10 gene, which was unreported previously. The variant did not significantly alter the expression of SOX10 at the mRNA level but the protein level. After the CHX treatment, the degradation of mutant SOX10 protein had slowed down. CONCLUSION The mutant SOX10 may affect the expression of downstream genes by affecting the degradation rate of its protein product.
Humans ; HEK293 Cells ; Mutation ; SOXE Transcription Factors/metabolism* ; Waardenburg Syndrome/genetics* ; Child

OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive soft-tissue sarcoma that commonly presents late in the disease due to its nonspecific symptoms. Due to its rarity, there is still no standardized treatment regimen. A multimodal approach remains to be the cornerstone of treatment-complete cytoreductive surgery, chemotherapy, and radiotherapy. Despite advances in treatment, use of targeted and immunotherapy, an improved survival rate remains to be elusive. This paper presents a case of DSRCT in a 21-year-old Filipino patient as well as a review of the related literature of reported DSRCT cases in females and the emerging therapies for the disease.