Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Objective To design and construct chimeric antigen receptor(CAR)T cells targeting Trop2,establish an in vitro cell exhaustion model through continuous antigen stimulation,and investigate their anti-tumor activity and exhaustion characteristics.Methods The second-generation CAR plasmid was constructed based on the single-chain variable fragment(scFv)sequence of Sacituzumab Govitecan targeting Trop2.The viral vector titer was determined by retroviral vector packaging and gradient dilution.Peripheral blood mononuclear cells(PBMCs)from healthy donors were isolated using Ficoll density gradient centrifugation,and CAR virus vectors were transduced into PBMCs activated with OKT-3/IL-2 to generate Trop2-targeted CAR T cells.CAR expression levels were assessed by flow cytometry using MYC tags.In vitro 3 tumor cell models were established,including human ovarian cancer cells(SKOV3),human breast cancer cells(MDA-MB-453),and human lung cancer cells(A549).The expression of the Trop2 antigen in these models was confirmed using flow cytometry.Additionally,luciferase assay was employed to evaluate the cytotoxic efficiency of Trop2-targeted therapy at various effector-to-target ratios.An in vitro CAR-T exhaustion model was developed,and the long-term killing ability of CAR-T cells was dynamically monitored using the Incucyte live-cell imaging system.The PD-1/TIM-3 phenotype of CAR-T cells was analyzed by flow cytometry,and cytokine secretion levels were quantified using the cytometric bead array(CBA).Transcriptomic sequencing and RT-qPCR were employed to validate the differentially expressed genes associated with exhaustion.Results The second-generation CAR T cells targeting Trop2 were successfully constructed.Compared to the P-T group,in vitro experiments demonstrated that these CAR T cells exhibited antigen-specific and dose-dependent cytotoxic effects against tumor cells with high Trop2 expression,such as MDA-MB-453 and SKOV3.A CAR-T cell exhaustion model established through repeated tumor antigen stimulation in vitro revealed that,compared to the initial state,the exhausted Trop2 CAR-T cells exhibited significantly reduced tumor-killing capacity while P-T cells showed almost no killing effect,the expression of inhibitory receptors(PD-1 and TIM-3)was up-regulated on the surface of exhausted CAR-T cells,and the secretion of effector cytokines was diminished.Transcriptomic analysis identified multiple differentially expressed genes in the exhausted CAR-T cells.Pathways related to immune response and T cell receptor signaling were down-regulated,while apoptosis-related pathways were activated.RT-qPCR further confirmed abnormal expression of immunoregulatory genes,including IL3,IL5,and IL13(P<0.05).Conclusion During continuous in vitro tumor antigen stimulation,the second-generation CAR-T cells targeting the Trop2 antigen demonstrate declined anti-tumor activity,weakened effector function and up-regulated expression of exhaustion-related molecules.

[摘 要] 目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建表达PD-1 shRNA的CD19 CAR分子基因，将其包装成逆转录病毒载体，通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞，获得三种CAR-T细胞，分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平，流式细胞术检测三种CAR-T细胞中PD-1表达水平，萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞（人淋巴瘤daudi细胞）的杀伤功能。结果：RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体，病毒载体拷贝数均高于1×107拷贝/mL，转导人原代T细胞获得CAR-T细胞，RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比，PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞，其对CD19阳性靶细胞的杀伤率显著提高，PD-1 mRNA及其翻译产物PD-1的表达减少，CAR-T细胞的耗竭减缓。

Objective: To investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). Methods: Anti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. Results: XBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. Conclusion The results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

Objective:To determine the incidence of adrenal incidentalomas(AIs) in patients with diabetes mellitus and the metabolism profiles.Methods:A total of 615 hospitalized patients with diabetes mellitus in the Department of Endocrinology and Metabolism of Peking University People′s Hospital from March 2020 to May 2021 were retrospectively included in this study. AIs were screened by unenhanced chest computed tomography(CT) retrospectively and subsequently confirmed by multiplanar reconstruction. Participants′ physical indicators, metabolic profiles, and adrenal function parameters were collected. Unpaired t test, Mann-Whitney U test, and Chi-Square test were adopted to compare the metabolism profiles between diabetes mellitus patients with or without AIs. Regression models were used to estimate the correlations between AIs and the metabolism profiles such as blood glucose, blood lipids, blood pressure, and the adrenal function parameters.Results:Twenty-seven out of 615 participants were detected with AIs(4.4%). Patients with AIs had higher body mass index, waist circumference, and hip circumference than patients without AIs [(29.4±5.1)kg/m 2vs(26.8±3.8)kg/m 2,P=0.018; (102.3±11.7)cm vs(95.8±10.3)cm, P=0.002; (107.3±10.1)cm vs(101.4±7.6)cm, P=0.008]. The levels of serum uric acid and urinary albumin/creatinine ratio were also significantly increased in patients with AIs [(409.6±118.1)μmol/L vs(357.4±100.6)μmol/L, P=0.009; 21.25(7.49, 180.24)mg/g vs 8.60(4.71, 34.56)mg/g, P=0.010]. Besides, individuals with AIs were also associated with a higher risk of co-existing hypertension( P=0.045). Conclusion:The incidence of AIs in patients with diabetes is 4.4%. The presence of AIs in patients with diabetes may associated with increased risk of obesity and hypertension.

Objective To explore EEG characteristics and the therapeutic effect in children with electrical status epilepticus during slow sleep(ESES).Methods The eligible ESES cases in our center from 2014 to 2020 were included.The age at diagnosis of ESES,the duration of ESES,spike wave index(SWI)during wakefulness and the distribution of spike wave during the period of ESES,age at seizure onset,the clinical syndromes and the outcomes after treatment were analyzed.The ESES cases were divided into 4 groups according to the distribution of spike wave:focal ESES,unilateral ESES,bilateral asymmetric ESES,multiple foci ESES.The SWI during the awake stage were divided into 3 groups based on the different rates:≤20%,21%～49%,≥50%.The therapeutic outcomes were classified into three groups:satisfactory response,seizure control and ineffective.Results 50 cases were included,with 32 males and 18 females.The average onset age of ESES was 6 years and 7 months,and the average duration of ESES was 28 months.A significant correlation between the distribution of ESES and the thera-peutic effects was found,bilateral asymmetric ESES had a good therapeutic effects,while multiple foci ESES showed a poor therapeutic effects.The duration of ESES was significantly correlated with therapeutic effects,and the efficacy was worse when the duration was longer than 1 year.A significant relationship between the SWI during wakefulness of ESES and the therapeutic effects was detected,the patient with SWI≤20%during wakefulness had a good therapeutic effect.There was a negative correlation between the onset age of ESES and the duration of ESES and SWI index during wakefulness.There was a positive correlation between the duration of ESES and SWI index during wakefulness.Conclusion Our results suggest that onset age,distribution,duration and SWI during wake-fulness of ESES were correlated with therapeutic outcomes,The patient with SWI≤20%during wakefulness had a good therapeutic effect and have unfavorable outcomes with ESES last more than 1 year.The earlier onset of ESES,the longer duration of ESES and higher SWI during wakefulness will be showed..

Objective:To explore the correlation between clinical phenotypes and genotypes among 46 children with SCN1A-related developmental epileptic encephalopathy (DEE). Methods:Clinical data of 46 children with DEE and SCN1A variants identified at the Guangzhou Women and Children′s Medical Center between January 2018 and June 2022 were collected. The children were grouped based on their age of onset, clinical manifestations, neurodevelopmental status, and results of genetic testing. The correlation between SCN1A genotypes and clinical phenotypes was analyzed. Results:Among the 46 patients, 2 children (4.35%) had developed the symptoms before 3 months of age, 42 (91.30%) were between 3 to 9 months, and 2 cases (4.35%) were after 10 months. Two cases (4.35%) presented with epilepsy of infancy with migrating focal seizures (EIMFS), while 44 (95.7%) had presented with Dravet syndrome (DS), including 28 cases (63.6%) with focal onset (DS-F), 13 cases (29.5%) with myoclonic type (DS-M), 1 case (2.27%) with generalized type (DS-G), and 2 cases (4.55%) with status epilepticus type (DS-SE). Both of the two EIMFS children had severe developmental delay, and among the DS patients, 7 cases had normal development, while the remaining had developmental delay. A total of 44 variants were identified through genetic sequencing, which included 16 missense variants and 28 truncating variants. All EIMFS children had carried the c. 677C>T (p.Thr226Met) missense variant. In the DS group, there was a significant difference in the age of onset between the missense variants group and the truncating variants group ( P < 0.05). Missense variants were more common in D1 (7/15, 46.7%) and pore regions (8/15, 53.3%), while truncating variants were more common in D1 (12/28, 42.9%). Children with variants outside the pore region were more likely to develop myoclonic seizures. Conclusion:The clinical phenotypes of DEE are diverse. There is a difference in the age of onset between individuals with truncating and missense variants in the SCN1A gene. Missense variants outside the pore region are associated with a higher incidence of myoclonic seizures.