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.

It is discovered that activated caspase-3 tends to induce apoptosis in gasdermin E (GSDME)-deficient cells, but pyroptosis in GSDME-sufficient cells. The high GSDME expression and apoptosis resistance of pancreatic ductal adenocarcinoma (PDAC) cells shed light on another attractive strategy for PDAC treatment by promoting pyroptosis. Here we report a hGLuc-hGSDME-PCA system for high-throughput screening of potential GSDME activators against PDAC. This screening system neatly quantifies the oligomerization of GSDME-N to characterize whether pyroptosis occurs under the stimulation of chemotherapy drugs. Based on this system, ponatinib and perifosine are screened out from the FDA-approved anti-cancer drug library containing 106 compounds. Concretely, they exhibit the most potent luminescent activity and cause drastic pyroptosis in PDAC cells. Further, we demonstrate that perifosine suppresses pancreatic cancer by promoting pyroptosis via caspase-3/GSDME pathway both in vitro and in vivo. Collectively, this study reveals the great significance of hGLuc-hGSDME-PCA in identifying compounds triggering GSDME-dependent pyroptosis and developing promising therapeutic agents for PDAC.

During coronavirus disease 2019 epidemic, the treatment of severe trauma has been impacted. The Consensus on emergency surgery and infection prevention and control for severe trauma patients with 2019 novel corona virus pneumonia was published online on February 12, 2020, providing a strong guidance for the emergency treatment of severe trauma and the self-protection of medical staffs in the early stage of the epidemic. With the Joint Prevention and Control Mechanism of the State Council renaming "novel coronavirus pneumonia" to "novel coronavirus infection" and the infection being managed with measures against class B infectious diseases since January 8, 2023, the consensus published in 2020 is no longer applicable to the emergency treatment of severe trauma in the new stage of epidemic prevention and control. In this context, led by the Chinese Traumatology Association, Chinese Trauma Surgeon Association, Trauma Medicine Branch of Chinese International Exchange and Promotive Association for Medical and Health Care, and Editorial Board of Chinese Journal of Traumatology, the Chinese expert consensus on emergency surgery for severe trauma and infection prevention during coronavirus disease 2019 epidemic ( version 2023) is formulated to ensure the effectiveness and safety in the treatment of severe trauma in the new stage. Based on the policy of the Joint Prevention and Control Mechanism of the State Council and by using evidence-based medical evidence as well as Delphi expert consultation and voting, 16 recommendations are put forward from the four aspects of the related definitions, infection prevention, preoperative assessment and preparation, emergency operation and postoperative management, hoping to provide a reference for severe trauma care in the new stage of the epidemic prevention and control.

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 screen new drugs for treatment of phenylalanine hydroxylase deficiency.Methods:From October 2019 to October 2020, virtual drug screening was performed in Center of Genetic Medicine, Nanjing Maternity and Child Health Care Hospital, Women's Hospital of Nanjing Medical University computer according to the characteristics of the binding ability of phenylalanine hydroxylase to drug spatial structure. Ten candidate drugs were screened from the FDA drug library (including 2 697 kinds of active pharmaceutical ingredients). A eukaryotic expression system was used to determine the effects of drugs on the activity of phenylalanine hydroxylase at the molecular level. Drug-sensitive mutants were screened.Results:Among the 10 candidate drugs, neoplasm hydrochloride, fluocinonide acetate and risperidone increased 23% [ t = 18.21, P < 0.001, vs. non-drug-treated phenylalanine hydroxylase group (i.e., only solvent and no drug added to the reaction system)], 21% ( t = 3.44, P < 0.05, vs. non-drug-treated phenylalanine hydroxylase group), 31% ( t = 19.57, P < 0.001, vs. non-drug-treated phenylalanine hydroxylase group) of the activity of phenylalanine hydroxylase. The remaining drugs exhibited weak even inhibitory effects on the activity of phenylalanine hydroxylase. 25% of p.D101N mutant could be activated by risperidone ( t = 15.86, P < 0.001, vs. non-drug-treated p.D101N mutant group). Conclusion:Neoplasm hydrochloride, fluocinonide acetate and risperidone can be used as potential therapeutic drugs for phenylalanine hydroxylase deficiency, and p.D101N mutant can be used as the drug-sensitive mutation site.

Objective:To investigate the clinical characteristics and pathogenic mutations of propionic acidemia.Methods:Clinical data of two patients with propionic acidemia admitted to the Obstetrics and Gynecology Hospital of Nanjing Medical University from May 2017 to June 2018 were collected. Genomic DNA was extracted from the peripheral blood of the patients and their parents. Inherited disease panel based on Ion Torrent semiconductor sequencing technology was performed to detect gene mutations, and those with suspected pathogenic mutations were verified by Sanger sequencing. Descriptive statistical analysis was used for data analysis.Results:Case 1 was suspected of sepsis and admitted to the Obstetrics and Gynecology Hospital of Nanjing Medical University due to "drowsiness and milk rejection" on the second day after birth. Tandem mass spectrometry suggested the level of propionyl carnitine and its ratios to acetylcarnitine and free carnitine were increased. Urine gas chromatography-mass spectrometry showed elevated 3-hydroxypropionic acid and methylcitric acid. Genetic analysis revealed that the infant carried c.331C>T (p.R111X)/c.1228C>T (p.R410W) compound heterozygous mutations in the PCCB gene. The infant was diagnosed with propionic acidemia and treated with a special diet with an L-Carnitine supplement but died of sudden coma and vomiting without precipitating factors at three months of age. Case 2 presented with sudden vomiting, drowsiness, and anergia on the admission at five-months old. Tandem mass spectrometry showed increased propionyl carnitine level and its ratios. Compound heterozygous mutations of c.146delG (p.G49EfsX16)/c.1253C>T (p.A418V) in the PCCB gene were identified in the patient, of which c.146delG (p.G49EfsX16) was a de novo mutation and was evaluated as a pathogenic mutation. The patient was on a special diet with an L-Carnitine supplement, but with disobedience. Followed up to the age of three years and eight months, the child was severely underdeveloped. Conclusions:Neonates with clinically suspected sepsis may have propionic acidemia, and tandem mass spectrometry and genetic testing should be performed as soon as possible to confirm or rule out the diagnosis. Further investigations on the pathogenesis and function of the new mutation are still needed.

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC GSDME-dependent pyroptosis.

Objective:To analyze the detection of neonatal inherited metabolic diseases in Nanjing.Methods:We researched the results of 175 767 newborns by tandem mass spectrometry from December 2013 to July 2018. Amino acids, acylcarnitines, and succinylacetone were detected by non-derivatized tandem mass spectrometry to screen the abnormity of newborn amino acid, organic acid, or fatty acid oxidation metabolism disease. Gene panels based on high throughput sequencing technology were carried out to detect gene mutation of positive neonates. Descriptive statistics were used to analyze all the data.Results:The positive rate of primary screening was 2.1% (3 691/175 767), 3 598 of 3 691 positive cases were recalled. At last, 62 cases of the inherited metabolic disease were diagnosed. Among them, there were 35 cases of amino acid metabolism disease, 12 cases of organic acid metabolism disorder, and 15 cases of fatty acid metabolism defect. The total incidence of neonatal inherited metabolic disease was 0.035 3%, among which amino acid metabolic diseases were 0.019 9%, organic acid metabolic diseases were 0.006 8%, and fatty acid metabolic diseases were 0.008 5%. The diseases with the highest incidence were phenylalanine hydroxylase deficiency (0.015 9%), methylmalonic acidemia (0.005 1%), and primary carnitine deficiency (0.005 1%). Among 62 children, 51 (82.2%) were diagnosed by gene diagnosis (including 17 cases of phenylalanine hydroxylase deficiency and 34 cases of other inherited metabolic diseases). Another 11 children with phenylalanine hydroxylase deficiency refused gene diagnosis. Two pathogenic mutations were found in 17 children with phenylalanine hydroxylase deficiency. Two pathogenic mutations were found in 29 of the other 34 children with inherited metabolic disease, which were from their parents, while only one pathogenic mutation was found in the other five children, of which two cases with hypermethioninemia were autosomal dominant inheritance.Conclusions:Neonatal inherited metabolic diseases with high incidence in Nanjing are phenylalanine hydroxylase deficiency, methylmalonic acidemia, and primary carnitine deficiency. Some cases screened by tandem mass spectrometry only showed abnormal screening indicators. No specific clinical symptoms were found during follow-up, and further follow-up was needed.

Objective:To examine the short-term outcomes of modified load-sharing rip-stop repair (mLSRS) and suture-bridge repair (SB) for large rotator cuff tear.Methods:The clinical data of patients who underwent shoulder arthroscopy for large rotator cuff repair at Department of Orthopedic, China Academy of Chinese Medical Sciences Wang Jing Hospital from January 2017 to December 2018 were collected. Totally 111 patients (39 in mLSRS group and 72 in SB group) were included in the study. There were 45 males and 66 females, aged (58.0±6.9)years (range: 40 to 72 years).A total of 39 pairs of patients were successfully matched by propensity score matching. The pain of shoulder joint was assessed by visual analogy score (VAS) , the shoulder function was assessed by Constant-Murley score, the abduction strength of bilateral shoulder joint was assessed by 90? force assessment method of upper limb on the plane of scapula, and the healing of rotator cuff on the operative side was assessed by sugaya ultrasonic classification standard. The difference of short-term outcome between the two groups were compared using t test, χ 2 test, Mann-Whitney U test, repeated measures design anova and multivariate analysis of variance. Results:At the 4th week after surgery, the VAS of mLSRS group was significantly lower than SB group(1 (2) vs. 2 (2) , F=5.32, P=0.02). One year after surgery, Constant-Murley score of two groups were significantly higher than those before surgery, but there was no significant difference between two groups(89.1±5.7 vs. 87.6±7.3, t=1.00, P=0.32). Abductor strength of affected limbs were lower than that of the opposite side, but there were no statistically significant differences between two groups(4.28±1.61 vs. 4.54±1.64, t=-0.70, P=0.49). According to the Sugaya classification, 32 cases in mLSRS group were healed while 7 cases were not healed,30 cases in SB group were healed while 9 cases were not healed(χ 2=0.32, P=0.58). Conclusion:The use of mLSRS fixation construct on large rotator cuff tear can reduce pain and the short-term outcome is reliable.