Objective:To assess the prevalence of pathogenic variants in the acid alpha-glucosidase ( GAA) gene among newborns in Nanjing and provide a reference for early screening, diagnosis, and treatment of Pompe disease. Methods:This retrospective study conducted on 30 043 live births at Women's Hospital of Nanjing Medical University (Nanjing Women and Children's Healthcare Hospital) from March 2022 to October 2024. Heel blood samples were collected within 48 h after birth to make dried blood spots. Chip-capture-based next-generation sequencing was used to detect pathogenic/likely pathogenic (P/LP) GAA variants. Suspected cases underwent Sanger sequencing validation and GAA enzyme activity assay to summarize the carrier status of pathogenic variants in the GAA gene among newborns. Descriptive statistical analysis was used. Results:Among the 30 043 newborns, 232 carriers (one P/LP variant) and four presumptive cases (two P/LP variants) were identified. The GAA activity of suspected cases 1 and 2 was normal, and the two variants were in cis, leading to a clinical diagnosis of carriers. Presumptive case 3 had a GAA activity of 0.17 μmol/(L·h), below the normal range [2.63-21.69 μmol/(L·h)]; the two variants were in trans, without clinical manifestations of Pompe disease follow-up to 2 years and 1 month, resulting in a clinical diagnosis of a potential patient. Presumptive case 4 had a GAA activity of 0.36 μmol/(L·h), below the normal range; the two variants were in cis, and two pseudodeficiency variants [c.1726G>A(p.G576S) and c.2065G>A(p.E689K)] were also found, leading to a final clinical diagnosis of a carrier. Therefore, a total of 235 carriers of P/LP GAA variants were identified, with a carrying rate of 1/128 (235/30 043), and one potential patient was identified with an incidence rate of 1/30 043. The top five common GAA variants were c.2132_2133delinsGG, c.503G>A, c.-32-13T>G, c.2662G>T, and c.2238G>C, with allele frequencies of 0.078% (47/60 086), 0.038% (23/60 086), 0.020% (12/60 086), 0.018% (11/60 086), and 0.017% (10/60 086), respectively. Protein structure prediction results showed that c.2132_2133delinsGG resulted in a shortened two β-sheet in GH31(β/α) 8 barrel catalytic domain and altered signal peptide and prepeptide conformation. c.503G>A would extend one β-sheet and add an additional β-sheet in the N-terminal domain. Conclusion:Newborn genetic screening combined with GAA activity measurement can exclude the interference of pseudodeficiency alleles, improve screening efficiency and accuracy, and provide a reference for the clinical diagnosis and genetic counseling of Pompe disease.

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Objective:To assess the prevalence of pathogenic variants in the acid alpha-glucosidase ( GAA) gene among newborns in Nanjing and provide a reference for early screening, diagnosis, and treatment of Pompe disease. Methods:This retrospective study conducted on 30 043 live births at Women's Hospital of Nanjing Medical University (Nanjing Women and Children's Healthcare Hospital) from March 2022 to October 2024. Heel blood samples were collected within 48 h after birth to make dried blood spots. Chip-capture-based next-generation sequencing was used to detect pathogenic/likely pathogenic (P/LP) GAA variants. Suspected cases underwent Sanger sequencing validation and GAA enzyme activity assay to summarize the carrier status of pathogenic variants in the GAA gene among newborns. Descriptive statistical analysis was used. Results:Among the 30 043 newborns, 232 carriers (one P/LP variant) and four presumptive cases (two P/LP variants) were identified. The GAA activity of suspected cases 1 and 2 was normal, and the two variants were in cis, leading to a clinical diagnosis of carriers. Presumptive case 3 had a GAA activity of 0.17 μmol/(L·h), below the normal range [2.63-21.69 μmol/(L·h)]; the two variants were in trans, without clinical manifestations of Pompe disease follow-up to 2 years and 1 month, resulting in a clinical diagnosis of a potential patient. Presumptive case 4 had a GAA activity of 0.36 μmol/(L·h), below the normal range; the two variants were in cis, and two pseudodeficiency variants [c.1726G>A(p.G576S) and c.2065G>A(p.E689K)] were also found, leading to a final clinical diagnosis of a carrier. Therefore, a total of 235 carriers of P/LP GAA variants were identified, with a carrying rate of 1/128 (235/30 043), and one potential patient was identified with an incidence rate of 1/30 043. The top five common GAA variants were c.2132_2133delinsGG, c.503G>A, c.-32-13T>G, c.2662G>T, and c.2238G>C, with allele frequencies of 0.078% (47/60 086), 0.038% (23/60 086), 0.020% (12/60 086), 0.018% (11/60 086), and 0.017% (10/60 086), respectively. Protein structure prediction results showed that c.2132_2133delinsGG resulted in a shortened two β-sheet in GH31(β/α) 8 barrel catalytic domain and altered signal peptide and prepeptide conformation. c.503G>A would extend one β-sheet and add an additional β-sheet in the N-terminal domain. Conclusion:Newborn genetic screening combined with GAA activity measurement can exclude the interference of pseudodeficiency alleles, improve screening efficiency and accuracy, and provide a reference for the clinical diagnosis and genetic counseling of Pompe disease.

BACKGROUND:Haploidentical hematopoietic stem cell transplantation is associated with a higher rate of graft rejection and therefore often requires a higher CD34+ cell dose,but the findings reported in existing studies regarding the relationship between CD34+ cell dose and study endpoints after allogeneic hematopoietic stem cell transplantation are controversial. OBJECTIVE:To investigate the effect of CD34+ cell dose on clinical outcomes of haploidentical hematopoietic stem cell transplantation for malignant hematological diseases. METHODS:135 patients who underwent haploidentical hematopoietic stem cell transplantation at Hematopoietic Stem Cell Transplantation Center,Department of Hematology,First Affiliated Hospital of Zhengzhou University between January 2019 and December 2021 were included.Combining the results of previous studies and our center's experience,the cohort was divided into two groups using a CD34+ cell count of 5.0×106/kg as the cut-off point.Clinical outcomes related to graft implantation,relapse incidence,non-relapse mortality,overall survival and progression-free survival were evaluated in both groups. RESULTS AND CONCLUSION:(1)CD34+ cell dose correlated with platelet engraftment,with platelets implanted earlier in the high-dose group than in the low-dose group(14 days vs.16 days,P=0.013).(2)There was no significant difference in 3-year overall survival between the two groups(67.5%vs.53.8%,P=0.257);nor was there a significant difference in progression-free survival between the two groups(65.6%vs.44.2%,P=0.106),but stratified analysis based on disease risk index revealed an association with elevated 3-year progression-free survival in the high-dose group among low-risk patients(72.0%vs.49.3%,P=0.036).(3)The cumulative 3-year relapse incidence was smaller in the high-dose group than in the low-dose group(16.0%vs.33.5%,P=0.05).(4)The rate of non-relapse mortality within 100 days was greater in the high-dose group than in the low-dose group,but there was no significant difference(17.3%vs.6.7%,P=0.070);stratified analysis revealed that non-relapse mortality within 100 days was significantly higher in the high-dose group than in the low-dose group(20.0%vs.3.3%,P=0.046).(5)In conclusion,CD34+ cell doses>5.0×106/kg promote early platelet implantation,improve 3-year progression-free survival in low-risk patients at transplantation and reduce the cumulative relapse incidence.However,in high-risk patients,high-dose CD34+ cells result in increased non-relapse mortality within 100 days after transplantation,which is considered to be possibly associated with an increased occurrence of severe acute graft versus host disease in the early post-transplantation period.Therefore,it is considered that graft versus host disease monitoring should be enhanced in patients who transfused high-dose CD34+ cells.

BACKGROUND:Allogeneic hematopoietic stem cell transplantation is an effective and even the only way to cure various hematological diseases,but the short-term mortality rate is relatively high after transplantation. OBJECTIVE:To investigate the risk factors affecting the overall survival of patients with hematological diseases in the short term(within 100 days)after allogeneic hematopoietic stem cell transplantation,so as to reduce mortality and effectively prevent related risks in the short term(within 100 days)after allogeneic hematopoietic stem cell transplantation. METHODS:Clinical data of 585 patients with hematological diseases who underwent allogeneic hematopoietic stem cell transplantation at the Hematopoietic Stem Cell Transplantation Center of First Affiliated Hospital of Zhengzhou University from January 1,2018 to June 30,2021 were retrospectively analyzed.The risk factors that affected overall survival within 100 days after allogeneic hematopoietic stem cell transplantation were explored. RESULTS AND CONCLUSION:A total of 585 patients with hematologic diseases underwent allogeneic hematopoietic stem cell transplantation.92 patients died within 100 days after transplantation,with a mortality rate of 15.7%(92/585).The median age of death cases was 26.5 years old(1-56 years),and the median survival time of death cases was 48 days(0-97 days).Univariate analysis exhibited that age≥14 years old,acute graft-versus-host disease,grade IV acute graft-versus-host disease,bacterial bloodstream infection,as well as carbapenem-resistant organism bloodstream infection,were risk factors for overall survival within 100 days after allogeneic hematopoietic stem cell transplantation(P<0.05).Multivariate regression analysis showed that age≥14 years old,grades Ⅲ-Ⅳ acute graft-versus-host disease,bacterial bloodstream infection,and carbapenem-resistant organism bloodstream infections were independent risk factors for overall survival(within 100 days)in patients after allogeneic hematopoietic stem cell transplantation.Hazard ratios were 1.77(95%CI 1.047-2.991),7.926(95%CI 3.763-16.695),2.039(95%CI 1.117-3.722),and 3.389(95%CI 1.563-7.347),respectively.In conclusion,all-cause mortality rate after allogeneic hematopoietic stem cell transplantation is relatively high in the short term.A timely diagnosis and effective treatment of bacterial bloodstream infection and acute graft-versus-host disease are essential to improving allogeneic hematopoietic stem cell transplantation outcomes.

Objective:To explore the pathogenicity and genotype-phenotype correlation of a c. 158G>A variant of phenylalanine hydroxylase ( PAH) gene among patients with PAH deficiency. Methods:Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed.Results:Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c. 158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c. 158G>A variants and a heterozygous c. 842+ 2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c. [158G>A, 842+ 2T>A]/c.728G>A and c. [158G>A, 842+ 2T>A]/c.611A>G, respectively, and one with c. [158G>A, c. 722G>A]/c.728G>A. The c. 158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. Conclusion:When the c. 158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

Objective:To investigate the value of implantable cardiac monitor (ICM) in the diagnosis and treatment of patients over 60 years old with unexplained syncope.Methods:This was a multi-center, prospective cohort study. Between June 2018 and April 2021, patients over the age of 60 with unexplained syncope at Beijing Hospital, Fuwai Hospital, Beijing Anzhen Hospital and Puren Hospital were enrolled. Patients were divided into 2 groups based on their decision to receive ICM implantation (implantation group and conventional follow-up group). The endpoint was the recurrence of syncope and cardiogenic syncope as determined by positive cardiac arrhythmia events recorded at the ICM or diagnosed during routine follow-up. Kaplan‐Meier survival analysis was used to compare the differences of cumulative diagnostic rate between the 2 groups. A multivariate Cox regression analysis was performed to determine independent predictors of diagnosis of cardiogenic syncope in patients with unexplained syncope.Results:A total of 198 patients with unexplained syncope, aged (72.9±8.25) years, were followed for 558.0 (296.0,877.0) d, including 98 males (49.5%). There were 100 (50.5%) patients in the implantation group and 98 (49.5%) in the conventional follow-up group. Compared with conventional follow-up group, patients in the implantation group were older, more likely to have comorbidities, had a higher proportion of first degree atrioventricular block indicated by baseline electrocardiogram, and had a lower body mass index (all P<0.05). During the follow-up period, positive cardiac arrhythmia events were recorded in 58 (58.0%) patients in the ICM group. The diagnosis rate (42.0% (42/100) vs. 4.1% (4/98), P<0.001) and the intervention rate (37.0% (37/100) vs. 2.0% (2/98), P<0.001) of cardiogenic syncope in the implantation group were higher than those in the conventional follow-up group (all P<0.001). Kaplan-Meier survival analysis showed that the cumulative diagnostic rate of cardiogenic syncope was significantly higher in the implantation group than in the traditional follow-up group ( HR=11.66, 95% CI 6.49-20.98, log-rank P<0.001). Multivariate analysis indicated that ICM implantation, previous atrial fibrillation, diabetes mellitus or first degree atrioventricular block in baseline electrocardiogram were independent predictors for cardiogenic syncope (all P<0.05). Conclusions:ICM implantation improves the diagnosis and intervention rates in patients with unexplained syncope, and increases diagnostic efficiency in patients with unexplained syncope.

Objective To evaluate the application value of golden angle radial sparse parallel(Grasp)sequence in contrast-enhanced MRI of liver.Methods The imaging data of 30 patients who underwent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid contrast-enhanced MRI of liver were collected.With the same equipment,images were collected by Grasp sequence and breath-hold sequence separately,with an interval of less than 3 months.The subjective and objective scores of the late arterial phase and portal venous phase images were evaluated.Results There were no significant differences in all subjective scores of the late arterial phase and portal venous phase images between the two sequences(P＞0.05).The signal-to-noise ratio(SNR)of the late arterial phase images in the Grasp sequence was lower than that in the breath-hold sequence(148.4±52.8 vs 195.6±68.4),and the difference was statistically significant(P＜0.01).Although the SNR of the portal venous phase in the Grasp sequence was lower than that in the breath-hold sequence,the difference was not statistically significant(P＞0.05).There was no statistical difference in the other objective scores between the two sequences(P＞0.05).Conclusion The image quality of Grasp sequence in contrast-enhanced MRI of liver can meet the diagnositic requirements,and it is suitable for patients with poor breath-hold capacity,which has important application value.

Objective:To explore the predictive value of secretogranin II (SCG2) for the prognosis of pancreatic neuroendocrine tumors (pNET) and to construct a nomogram prediction model based on SCG2.Methods:One hundred and thirty-three patients with pNET who underwent radical surgery at the Fourth Hospital of Hebei Medical University from August 2013 to December 2021 were retrospectively collected. The cohort, including 60 males and 73 females with a mean age of (53.70±12.08) years, was divided into a training set ( n=93) and a validation set ( n=40). Clinical data such as gender, age, tumor size, lymph node metastasis, TNM stage, liver metastasis, surrounding tissue invasion, tumor histological grade, and carcinoembryonic antigen (CA) 19-9 levels were gathered. Immunohistochemical staining was performed to detect SCG2 expression. Patient survival information was obtained through outpatient records or telephone follow-up. The Cox proportional hazard model was used to analyze prognostic factors. A nomogram was created based on the multivariate Cox regression analysis results. The calibration curve and the area under the receiver operating characteristic (ROC) curve were used to evaluate the nomogram's accuracy and discrimination. Results:The SCG2 expression in pNET patients with AJCC stage Ⅲ-Ⅳ was higher than in those with stage Ⅰ-Ⅱ. Similarly, patients with histological grade G3 had higher SCG2 levels compared to those with grades G1-G2. SCG2 expression was also elevated in patients older than 60 years compared to those 60 years or younger, and in patients with CA19-9>30 U/L compared to those with CA19-9≤30 U/L. These differences were all statistically significant (all P<0.05). Multivariate Cox regression analysis revealed that lymph node metastasis ( HR=3.132, 95% CI: 1.212-8.096, P=0.017), liver metastasis ( HR=2.685, 95% CI: 1.002-7.192, P=0.049), histological grade G3 ( HR=3.692, 95% CI: 1.229-11.088, P=0.011), and high SCG2 expression ( HR=52.181, 95% CI: 38.476-108.118, P=0.002) were associated with significantly higher risks of shorter disease free survival. Additionally, patients with longer tumor diameters ( HR=1.297, 95% CI: 1.088-1.545, P=0.004), histological grade G3 ( HR=19.625, 95% CI: 5.276-88.634, P<0.001), and high SCG2 expression ( HR=39.454, 95% CI: 17.317-97.263, P<0.001) had a higher risk of shorter overall survival. A nomogram prediction model was constructed using these above factors. The calibration curve demonstrated good alignment between predicted and actual outcomes in both the training and validation sets. The areas under the ROC curves for the disease-free survival and overall survival nomogram models for predicting 1-, 3-, and 5-year survival of pNET patients in both sets were above 0.8. Conclusion:High SCG2 expression is an independent risk factor for poor prognosis in pNET patients. The nomogram model based on SCG2 has high predictive efficacy for pNET patient prognosis.

Objective: To compare the clinical outcomes and biomechanical characteristics of 1-, 2-, and 3-level pedicle subtraction osteotomy (PSO), and establish selection criteria based on preoperative radiographic parameters. Methods: Patients undergone PSO to treat ankylosing spondylitis from February 2009 to May 2019 in Sun Yat-sen Memorial Hospital of Sun Yat-sen University were enrolled. According to the quantity of osteotomy performed, the participants were divided into group A (1-level PSO, n = 24), group B (2-level PSO, n = 19), and group C (3-level PSO, n = 11). Clinical outcomes were assessed before surgery and at the final follow-up. Comparisons of the radiographic parameters and quality-of-life indicators were performed among and within these groups, and the selection criteria were established by regression. Finite element analysis was conducted to compare the biomechanical characteristics of the spine treated with different quantity of osteotomies under different working conditions. Results: Three-level PSO improved the sagittal parameters more significantly, but resulted in longer operative time and greater blood loss (p < 0.05). Greater stress was found in the proximal screws and proximal junction area of the vertebra in the model simulating 1-level PSO. Larger stress of screws and vertebra was observed at the distal end in the model simulating 3-level PSO. Conclusion Multilevel PSO works better for larger deformity correction than single-level PSO by allowing greater sagittal parameter correction and obtaining a better distribution of stress in the hardware construct, although with longer operation time and greater blood loss. Three-level osteotomy is recommended for the patients with preoperative of global kyphosis > 85.95°, T1 pelvic angle > 62.3°, sagittal vertical alignment > 299.55 mm, and pelvic tilt+ chin-brow vertical angle > 109.6°.