Objective:To understand the regional prevalence and hotspot mutations through analysis of genetic screening results for newborns with pseudohypertrophic muscular dystrophy.Methods:A total of 22 813 newborns (12 065 males and 10 748 females) born at the Women's Hospital of Nanjing Medical University from March 18, 2022, to October 31, 2023, were selected. The Dystrophin gene ( DMD) was detected using chip capture next-generation sequencing technology, followed by bioinformatics analysis. Pathogenic mutations identified were validated using multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. Serum creatine kinase levels were also tested in suspected male patients. Descriptive analysis was applied for this study. Results:Among the 10 748 girls, 14 carriers of DMD gene were detected (0.013%), of which, nine cases were validated in the family; one case was a de novo mutation, five were inherited from the mother, and three were inherited from the father. The screening identified nine suspected patients among the boys (0.075%), and eight of them were confirmed by family validation, in which three were de novo mutations, and five were inherited from the mother. Among all identified DMD mutations, deletions were the most common one, accounting for 52.2% (12/23), incluling four cases of deletion at 49-51 exon. Conclusions:Newborn genetic screening based on chip capture next-generation sequencing technology combined with bioinformatics analysis is helpful in early detection of patients and carriers of pseudohypertrophy muscular dystrophy. According to the preliminary statistics, the incidence rate of DMD/BMD in this area is 1/1 341 male infants and the hotspot mutation is exon 49 to 51 deletion.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular genetic disorder characterized by progressive muscle atrophy, with most patients succumbing to heart or respiratory failure around the age of 20. This article systematically reviews the discovery, pathological research, diagnosis, and development of newborn screening for DMD. Since the 19th century, pioneers such as Bell, Conte, Meryon, Duchenne, and Gowers have laid the foundation for understanding this disease through their discoveries and descriptions of DMD. In the 1980s, molecular biology research further elucidated the pathological mechanisms of DMD and established diagnostic methods. Since the 1970s, newborn screening for DMD has flourished, evolving through various stages including creatine kinase testing, muscle-type creatine kinase isoenzyme testing, and genetic screening. With ongoing research, early screening and diagnostic protocols for DMD have been continuously refined and gradually implemented in clinical practice.

Objective:To establish the cut-off value of tetradecenoyl carnitine (C14∶1)/dodecenoyl carnitine(C12∶1) based on non-derivatized tandem mass spectrometry (MS/MS), and to explore the application value of C14∶1/C12∶1 to screen newborns for very long chain acyl-CoA dehydrogenase deficiency (VLCADD), determining the best combination of indicators for screening VLCADD.Methods:This retrospective study included data from 17 newborns with VLCADD detected by MS/MS and confirmed by acyl-CoA dehydrogenase very long chain ( ACADVL) gene detection, and 423 507 newborns with normal MS/MS results. The data from these newborns were collected from January 2014 to December 2021 as the newborns received neonatal screening in Nanjing Neonatal Disease Screening Center and Suzhou Neonatal Disease Screening Center. All newborns were divided into 3 groups: all newborns group, full-term newborns group and normal-birth-weight newborns group, and the cut-off values of C14∶1/C12∶1 for VLCADD in these 3 groups were determined by their receiver operating characteristic (ROC) curves individually. With these results, a total of 5 interpretation schemes were composed using different indicators alone or jointly: scheme 1 being C14∶1/C12∶1, scheme 2 being C14∶1, scheme 3 being C14∶1+C14∶1/C2+C14∶1/C16, scheme 4 being C14∶1/C12∶1+C14∶1, and scheme 5 being C14∶1/C12∶1+C14∶1+C14∶1/C2+C14∶1/C16. The detection rate, false-positive rate and positive predictive value of each scheme were calculated, and their screening efficiencies were statistically compared by Chi-square tests. Results:The cut-off values of C14∶1/C12∶1 for VLCADD in the 3 newborn groups were all 2.80. The detection rates of VLCADD with all 5 interpretation schemes were 17/17. Scheme 1 had the highest false positive rate [26.15‰ (11 075/423 524)] and the lowest positive predictive value [0.15% (17/11 092)]. Scheme 4 (Scheme 5) had the lowest false positive rate [0.02‰ (10/423 524)] and the highest positive predictive value [62.96% (17/27)]. Comparing scheme 4 (Scheme 5) with scheme 1, scheme 2 and scheme 3, the differences of false positive rate (χ2=302.30,11 191.50,32.06) and positive predictive value (χ2=102.51,3 485.61,13.83) were statistically significant (all P<0.001). Conclusion:C14∶1/C12∶1 was an effective auxiliary interpretive indicator for VLCADD in newborn screening, and the combination of C14∶1/C12∶1+C14∶1 was tested to be the best indicator for VLCADD screening based on non-derivatized tandem mass spectrometry.

Previous studies have revealed that patients with hypertrophic cardiomyopathy (HCM) exhibit differences in symptom severity and prognosis, indicating potential HCM subtypes among these patients. Here, 793 patients with HCM were recruited at an average follow-up of 32.78 ± 27.58 months to identify potential HCM subtypes by performing consensus clustering on the basis of their echocardiography features. Furthermore, we proposed a systematic method for illustrating the relationship between the phenotype and genotype of each HCM subtype by using machine learning modeling and interactome network detection techniques based on whole-exome sequencing data. Another independent cohort that consisted of 414 patients with HCM was recruited to replicate the findings. Consequently, two subtypes characterized by different clinical outcomes were identified in HCM. Patients with subtype 2 presented asymmetric septal hypertrophy associated with a stable course, while those with subtype 1 displayed left ventricular systolic dysfunction and aggressive progression. Machine learning modeling based on personal whole-exome data identified 46 genes with mutation burden that could accurately predict subtype propensities. Furthermore, the patients in another cohort predicted as subtype 1 by the 46-gene model presented increased left ventricular end-diastolic diameter and reduced left ventricular ejection fraction. By employing echocardiography and genetic screening for the 46 genes, HCM can be classified into two subtypes with distinct clinical outcomes.

OBJECTIVE: To investigate the correlation between serum vitamin D level and clinical outcomes after repair of rotator cuff tears. METHODS: A total 122 patients who met the inclusion criteria and were followed up for 12 months from March 2018 to March 2020 were analyzed retrospectively, including 50 males and 72 females with an average age of(62.10±8.39) years old (ranged, 34 to 82 years old). All patients were divided into deficiency group(vitamin D<20 μg/L) and control group(vitamin D≥20 μg/L), including 62 cases in the deficiency group, with vitamin D (14.80±3.18) μg/L;60 cases in the control group, with vitamin D(25.17±5.64) μg/L. The two groups were compared in terms of age, gender, body mass index(BMI), tear size, degree of retraction, degree of fatty infiltration, injury factors, postoperative pain VAS score, postoperative shoulder joint function score, and re-tear rate. The age of all patients was divided into two categories:<60 years old and ≥60 years old;BMI was divided into <24 kg/m² and ≥ 24 kg/m²;tear size was divided into ≤3 cm and >3 cm;retraction degree was divided into ≤2 cm and >2 cm;fat infiltration was divided into ≤2 grade and >2 grade;and the course of the disease was ≤3 months and >3 months. The correlation between Sugaya re-tear type and the variables listed above were analyzed and calculated. RESULTS: There were no major complications such as joint infection, anchor withdrawal and revision surgery in any of the 122 patients who were followed up on. There were no statistical differences in age, gender, injury factor, BMI, tear size, degree of retraction, degree of fatty infiltration, and duration of disease between the two groups (P>0.05). The Constant-Murley scores, UCLA scores, and ASES scores of the two groups all improved considerably after surgery(P<0.05);however, there was no statistical differences in the postoperative shoulder function scores between the two groups (P>0.05). There was significant difference in VAS between the two groups 1 month and 3 months after operation, with vitamin D deficiency group scoring higher, and there was no significant differences 6 and 12 months after operation. Tear size(>3 cm), degree of retraction(>2 cm), degree of fatty infiltration(>2 degree) were all shown to be the independent risk factors for retear after surgery by Logistic regression analysis(P<0.05). The comparison between the two groups of patients using a 2×5 row list showed that the Sugaya classification of rotator cuff re-tear(grade Ⅰto Ⅴ) between the vitamin D deficiency group and the control group was statistically different(t=14.228, P=0.007). It was discovered that the Sugaya classification after surgery was statistically different between the two groups. CONCLUSION Vitamin D deficiency is not correlated with clinical function scores and re-tear rate, however it is associated with the early postoperative pain (1 and 3 months) and the quality of rotator cuff healing (Sugaya classification) after surgery.
Adult ; Aged ; Aged, 80 and over ; Arthroscopy ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Rotator Cuff Injuries/surgery* ; Treatment Outcome ; Vitamin D

OBJECTIVE: To analyze the clinical features and genetic variants in four neonates with very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. METHODS: Neonates with a tetradecenoylcarnitine (C14:1) concentration at above 0.4 μmol/L in newborn screening were recalled for re-testing. Four neonates were diagnosed with VLCAD deficiency by MS-MS and genetic testing, and their clinical features and genotypes were analyzed. RESULTS: All cases had elevated blood C14:1, and the values of first recalls were all lower than the initial test. In 2 cases, the C14:1 had dropped to the normal range. 1 case has remained at above 1 μmol/L after the reduction, and the remainder one case was slightly decreased. In total eight variants of the ADACVL genes were detected among the four neonates, which included 5 missense variants and 3 novel variants (p.Met344Val, p.Ala416Val, c.1077+6T>A). No neonate showed salient clinical manifestations. CONCLUSION Above findings have enriched the spectrum of ADACVL gene mutations and provided a valuable reference for the screening and diagnosis of VLCAD deficiency.
Acyl-CoA Dehydrogenase/genetics* ; Acyl-CoA Dehydrogenase, Long-Chain ; Congenital Bone Marrow Failure Syndromes ; Genetic Testing ; Humans ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; Mitochondrial Diseases ; Muscular Diseases ; Tandem Mass Spectrometry

Objective:To identify the possible pathogenesis of a neonate with carnitine palmitoyltransferase 1A (CPT1A) deficiency by analyzing genetic variants.Methods:Potential variants were detected with an Ion Torrent semiconductor sequencer using a gene panel for inherited diseases, and candidate variants were verified by Sanger sequencing.Results:Genetic testing indicated that the neonate has carried c. 1895T>A(p.Leu632X) and c. 1153G>A(p.Ala385Thr) compound heterozygous variants of the CPT1A gene, which were inherited from his father and mother, respectively. Both variants were verified as novel through the retrieval of HGMD database, ClinVar database and literature. According to the standards and guidelines of the American College of Medical Genetics and Genomics, the c. 1895T>A variant was predicted as pathogenic(PVS1+ PM2+ PP4) and c. 1153G>A as likely pathogenic (PM1+ PM2+ PM3+ PP3). Conclusion:The c. 1895T>A and c. 1153G>A compound heterozygous variants of the CPT1A gene might underlie the pathogenesis in this child. Above results have provided a basis for clinical diagnosis and genetic counseling, and enriched the variant spectrum of the CPT1 deficiency.

Objective:To investigate the dynamic expression of histone methyltransferase (enhance of zeste homolog 2, EZH2) in peripheral blood B lymphocytes (CD19 +B) and memory B lymphocytes (CD19 +CD27 +B) of septic patients and its value in predicting prognosis in sepsis. Methods:From June 2018 to January 2020, 48 septic patients in the Intensive Care Unit of Shanghai East Hospital were enrolled, and 40 healthy adult volunteers were recruited as healthy controls. Septic patients were divided into the non-survivors (18 cases) and the survivors (30 cases) according to whether the patients survived at 28 days. Blood samples were collected at day 1, 3 and 7, blood routine, IL-6 and blood gas analysis were collected, and SOFA and APACHE Ⅱ scores were counted. Flow cytometry was used to detect the positive rate and the mean fluorescence intensity of EZH2 on CD19 +B lymphocytes, and the positive rate of EZH2 on CD19 +CD27 +B lymphocytes at different time points. In the healthy controls, fasting was taken only once in the morning. ROC curve was drawn and the area under the curve (AUC) was calculated to evaluate the value of expression of EZH2 on CD19 +B lymphocytes and CD19 +CD27 +B lymphocytes in predicting the prognosis of septic patients. Results:(1) Compared with the healthy controls, the positive rate and average fluorescence intensity of EZH2 on CD19 +B lymphocytes and the positive rate of EZH2 expression on CD19 +CD27 +B lymphocytes were significantly increased at day 1, 2 and 3 in septic patients ( P<0.05). Over time, the expression of EZH2 in CD19 +B lymphocytes and CD19 +CD27 +B lymphocytes increased gradually ( P<0.05). (2) Compared with the survivors, the positive rate of EZH2 on CD19 +B lymphocytes of the non-survivors was increased at day 1, but the positive rate of EZH2 on CD19 +CD27 +B lymphocytes of the non-survivors was decreased at day 3 and 7 ( P<0.05). (3) The positive rate of EZH2 on CD19 +B lymphocytes, APACHE Ⅱ score, SOFA score and IL-6 level in septic patients at day 1 were independently associated with 28-day mortality. (4) The AUC of APACHEⅡ score was 0.907 (95% CI: 0.825-0.990), and the sensitivity and the specificity were 88.89% and 76.67%. The AUC of SOFA score was 0.831 (95% CI: 0.706-0.955), and the sensitivity and the specificity was 66.67% and 86.67%; The AUC of EZH2 positive rate on CD19 +B lymphocytes were 0.799 (95% CI: 0.657-0.941), and the sensitivity and specificity were 88.89% and 80.77%, respectively, the sensitivity was better than SOFA score, and the specificity was higher than APACHEⅡ score. Conclusions:The high expression of EZH2 on B lymphocytes in septic patients is associated with poor prognosis. Dynamic monitoring of EZH2 expression on B lymphocytes has certain predictive value for sepsis.

BACKGROUND: The efficacy of entecavir (ETV) add-on peg-interferon therapy compared with ETV monotherapy in treatment-naïve hepatitis B virus (HBV) patients remains controversial. We investigated whether adding peg-interferon to ongoing ETV treatment leads to a better curative effect or not. METHODS: All patients have been recruited between August 2013 and January 2015 from the Shanghai Public Health Clinical Center and Zhongshan Hospital (China). Eligible HBV patients (n = 144) were randomly divided (1:1) to receive either ETV monotherapy (n = 70) or peg-interferon add-on therapy from week 26 to 52 (n = 74). Patients were followed-up for at least 2 years. Indexes including hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) seroconversion rate, sustained virologic response, transient elastography value, and histological scores were evaluated every 3 months until the end of the study. The rate of patients with HBsAg loss was defined as the primary endpoint criteria. RESULTS: At week 26, no patient achieved HBsAg seroconversion in either group. At week 52, one patient in the monotherapy group was HBsAg-negative but there was none in the combination therapy group. The monotherapy group showed significantly better liver function recovery results than the combination therapy group. At week 78, one patient in the combination group had HBsAg seroconverted. At week 104, only three patients in the combination therapy group were HBsAg-negative compared with one patient in monotherapy. The mean alanine aminotransferase and aspartate aminotransferase levels and transient elastography values decreased significantly compared with baseline. Both groups showed a favorable decrease in alpha-fetoprotein (monotherapy: 4.5 [2.8, 7.1] vs. 2.2 [1.8, 3.1] ng/mL, P < 0.001; combination therapy: 5.7 [3.0, 18.8] vs. 3.2 [2.0, 4.3] ng/mL, P < 0.001) and an improved result of liver biopsy examination scores. The combination group showed a better improvement in histology compared with the monotherapy group (mean transient elastography value 6.6 [4.9, 9.8] vs. 7.8 [5.4, 11.1] kPa, P = 0.028). But there was no significant difference in HBsAg conversion rate (1.8% [1/56] vs. 4.1% [3/73], P = 0.809) and HBeAg conversion rate (12.5% [7/56] vs. 11.0% [8/73], P = 0.787), as well as HBV-DNA, sustained virologic response (93.2% vs. 98.5%, P = 0.150) between the two groups. CONCLUSIONS: Both therapies supported liver function recovery and histology improvement. Combination therapy did not show better anti-viral efficacy in HBsAg or HBeAg seroconversion compared with monotherapy. However, combination therapy played a more positive role in reversing hepatic fibrosis compared with monotherapy. TRIAL REGISTRATION ClinicalTrials.gov: NCT02849132; https://clinicaltrials.gov/ct2/show/NCT02849132.

Objective:To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells (CMECs) in sepsis.Methods:Primary rat CMECs were isolated and cultured in vitro, and the cells in the logarithmic growth phase were used for experiments. Tetramethylazozolium colorimetry (MTT) was used to screen the safe and effective concentrations of paeoniflorin at 10, 20, and 40 μmol/L. The cells were divided into blank control group, lipopolysaccharide (LPS) group and low, medium and high concentration paeoniflorin pretreatment group. The cells in the blank control group were cultured in complete medium; the cells in the LPS group were challenged with LPS (1 mg/L) in complete medium; and the cells in the paeoniflorin pretreatment groups were pretreated with 10, 20, and 40 μmol/L paeoniflorin at 4 hours before LPS stimulation. The cells in each group were further cultured for 24 hours after LPS stimulation. The horseradish peroxidase (HRP) method was used to detect the permeability of rat CMECs. The enzyme-linked immunosorbent assay (ELISA) was used to detect the CXC chemokine ligand (CXCL1, CXCL2) levels in the cell supernatant. The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells. Western Blot was used to detect phosphorylated Src (p-Src), vascular endothelial-cadherin (VE-cadherin) and phosphorylated mitogen activated protein kinase (p-MAPK). Results:Compared with the blank control group, the permeability of rat CMECs in the LPS group was significantly increased. The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations, and the improvement was most obvious in the 40 μmol/L paeoniflorin group, with statistically significant difference as compared with the LPS group ( A value: 1.61±0.07 vs. 2.13±0.06, P < 0.01). ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group. However, the secretion of CXCL1 and CXCL2 in the cell supernatant was increased significantly under the induction of LPS. After pretreatment with paeoniflorin at different concentrations, the secretion of CXCL1 and CXCL2 in the cell supernatant was significantly reduced. The most obvious inhibitory effect on CXCL1 was 40 μmol/L paeoniflorin, and the most obvious inhibition on CXCL2 was 20 μmol/L paeoniflorin, the differences were statistically significant as compared with the LPS group [CXCL1 (ng/L): 337.51±68.04 vs. 829.86±65.06, CXCL2 (ng/L): 4.48±0.11 vs. 9.41±0.70, both P < 0.01]. RT-qPCR results showed that the mRNA expressions of CXCL1 and CXCL2 in the rat CMECs were consistent with the ELISA results. LPS could increase mRNA expressions of CXCL1 and CXCL2 in the rat CMECs, and pretreatment with different concentrations of paeoniflorin could significantly reduce the mRNA expressions of CXCL1 and CXCL2. The 40 μmol/L paeoniflorin had the best inhibitory effect on CXCL1 mRNA expression, and the 20 μmol/L paeoniflorin had the best inhibitory effect on CXCL2 mRNA expression, the differences were statistically significant as compared with the LPS group [CXCL1 mRNA (2 -ΔΔCt): 0.543±0.004 vs. 0.812±0.089, CXCL2 mRNA (2 -ΔΔCt): 10.52±0.71 vs. 17.68±1.09, both P < 0.01]. Western Blot results showed that moderate amounts of p-Src, VE-cadherin and p-MAPK proteins were expressed in the rat CMECs in the blank control group. After LPS stimulation, the expressions of p-Src and p-MAPK proteins were increased significantly, while the expression of VE-cadherin protein was decreased significantly. After pretreatment with different concentrations of paeoniflorin, the expressions of p-Src and p-MAPK proteins in the cells were decreased to varying degrees, while the expression of VE-cadherin protein was increased, and 40 μmol/L paeoniflorin had the most obvious effect, the differences were statistically significant as compared with the LPS group [p-Src protein (p-Src/GAPDH): 1.02±0.09 vs. 1.29±0.05, p-MAPK proteins (p-MAPK/GAPDH): 0.24±0.02 vs. 0.62±0.02, VE-cadherin protein (VE-cadherin/GAPDH): 0.64±0.03 vs. 0.31±0.02, all P < 0.01]. Conclusion:Paeoniflorin can regulate the Src/VE-cadherin pathway in CMECs, inhibit the expression and secretion of inflammation-related proteins and chemokines, and improve the cell permeability of CMECs induced by LPS.