Objective To assess the detection of urinary neutrophil gelatinase associated lipocalin (NGAL) and blood prohibitin (PHB) levels in early diagnosis of children with acute kidney injury (AKI).Methods One hundred and twenty children with severe allergic purpura,sepsis,kidney disease or heart disease admitted from June 2011 to June 2013 in our hospital were enrolled,including 60 cases with AKI and 60 cases without AKI;and 60 healthy children were selected as the control group.The urinary NGAL and blood PHB levels were measured with ELISA method.Results At d1 after diagnosis,the urinary NGAL level in severe AKI group [(146.76 ±61.22) μg/L] was higher than that in non-AKI group [(21.79 ± 17.31) μg/L] and control group [(17.42 ± 13.11) μg/L] (t =15.430 and 22.216,P ＜ 0.01).At d2 after diagnosis,the urinary NGAL level in severe AKI group [(82.31 ± 44.76) μg/L] was higher than that in non-severe AKI group [(21.56 ± 28.56) μg/L] (t =8.863,P ＜0.01).At d1 of diagnosis,the blood PHB level in severe AKI group was higher than that in non-severe AKI group [(14.03 ±6.43) vs.(8.01 ± 6.13),t =11.271,P =0.004];blood PHB in severe AKI group was higher than that in non-AKI group [(10.63 ± 4.21) vs.(8.00 ± 4.76),t =7.051,P =0.017].The levels of urinary NGAL and serum PHB gradually decreased over time in children with severe AKI.The area under the ROC curve (AUC) of urinary NGAL and blood PHB for diagnostic of AKI were 0.833 and 0.952 (P ＜ 0.01),respectively.The diagnostic rate of the combination of the two parameters was 100％.Conclusions The diagnostic value of PHB alone or NGAL in AKI still may be improved.The detection of PHB combined with NGAL can make up each other and improve the diagnosis of AKI,which contribute to the early diagnostic of AKI for clinical workers and provide effective intervention measures,and then reduce the mortality.

Objective To isolate and culture the rat limbal stem cells ( LSCs) in vitro,and investigate briefly their capacity for transdif-ferentiation into neural stem cells ( NSCs) with cytokine EGF,bFGF and RA.Methods LSCs derived from rats were cultured and identified by immunohistochemistry in vitro.LSCs were induced to differentiate into NSCs in the presence of EGF (20 ng/mL) ,bFGF (10 ng/mL) and with or without of RA(group 1 or group 2)(25 ng/mL) for seven days.Cultures without factors were used as control group.Then the neural marker Nestin of the coultured cells were measured by immunohistology staining.Furthermore,the positive cell rate was counted under micro-scope between the 2 groups and analyzed by statistical software.Results It showed that P63 was positive in LSCs.Nestin in both of the dif-ferentiation groups was positive at the rate of (77.01 ±6.32)%and (84.01 ±5.43)%respectively,of which the second group was higher than the first one (P<0.05).However,it was negative in the control group.A band of Nestin protein from cells was detected by western blot assay.Conclusion LSCs are successfully isolated and cultured in vitro.LSCs could be induced to differentiate into NSCs in the presence of EGF and bFGF.Moreover,the differentiation capability is enhanced in the condition of RA.

Twelve patients with maple syrup urine disease were treated with restricted natural protein intake,special nutrition powder,and vitmin B1.The manifestations of these patients were greatly improved after treatment.The blood levels ofleucine and valine were significantly decreased after treatment(P＜0.01).Although the level of branched-chain α-ketoacids in urine was reduced,but not significantly.

Objective To investigate the acid α glucosidase(GAA)gene mutations and clinical features of a Chinese patient exhibiting signs and symptoms of infantile glycogen storage disease type Ⅱ(GSD Ⅱ). Methods Clinical features of the patient were reviewed,and GAA activity in the patient＇s and her parents＇ whole leukocytes were measured. GAA coding regions were amplified by polymerase chain reaction(PCR),and analyzed by direct DNA sequencing. Results The patient showed feeding difficulties,generalized hypotonia and weakness starting at 2 months of age. Cardiomegaly and cardiomyopathy were found at 4 months. She died of cardiorespiratory failure at the age of 6 months. GAA activity in leukocytes was low in the patient(17.3% of the median normal range). Genotyping revealed the patient was a heterozygote for a novel nonsense mutation p.W738X and a previously reported nonsense mutation p.E888X. The reported pseudodeficiency allele c.1726G ＞ A;2065G ＞ Awas found in the patient and her mother. Conclusions Correct diagnosis was made for this patient by combination of GAA activity assay and genetic analysis. From the clinical course,this patient should be classified as infantile type of GSD Ⅱ,suggesting that the novel mutation p.W738X may have a damaging effect on the function of GAA. Pseudodeficiency allele found in this family highlights the importance of genetic analysis of GAA when performing diagnosis and prenatal diagnosis for the affected families,as this allele causes low GAA activity in normal individuals.

Objective To investigate the current distribution of hepatitis C virus (HCV) genotype in Southern China and to understand the HCV transmission and to infer its transmitting trend.Methods The HCV gene subtypes of 3 524 specimens from Southern China were detected and analyzed by polyonerase chain reaction (PCR)-fluorescence probe method or sequencing.The regular nested PCR and sequencing were used for the phylogenetic tree analysis when the fluorescence PCR inefficiently identifying virus isolates.Results Among 3 524 specimens,there were 2 922 cases from Guangdong,78 cases from Fujian,152 cases from Hainan and 372 cases from Guangxi.Genotype 1b comprised the majority (1 808/ 3 524,51.3％),followed by genotype 6a (925/3 524,26.2％),2a (298/3 524,8.46％),3a (246/ 3 524,6.98％),3b (200/3 524,5.68％) and 1a (27/3 524,0.77％).In addition,1 case wasgenotype 6e,1 case was genotype 6q,1 case was genotype 6r,3 case were genotype 6w,2 case were genotype 6xa,2 case were genotype 6n,and 1 case was genotype 6 with unclassified subtype.The genotype 1b accounted for the majority in most areas of 21 cities and counties in Guangdong Province,followed by genotype 6a.But in some areas,the major genotype was genotype 6a,followed by 1b.Genotype 4,genotype 5 and genotype 7 were not found in this study.Conclusions In the past two years,genotype 1b and 6a are still the epidemic genotypes in Guangdong,Guangxi and Hainan provinces.However,genotype 6a has replaced 1b as the dominant one in some areas in Guangdong Province.The distributions of HCV genotypes do not change significantly in Guangxi and Fujian provinces.

Objective:To analyze the value of droplet digital polymerase chain reaction (ddPCR) in detecting epidermal growth factor receptor (EGFR) mutations in peripheral blood circulating tumor DNA (ctDNA) of patients with non-small cell lung cancer (NSCLC).Methods:Peripheral blood samples of 63 patients with NSCLC who were treated in Shanxi Provincial Cancer Hospital from August 2018 to March 2019 were collected, and EGFR sensitive mutations in peripheral blood of patients were detected by ddPCR, and the results were compared with the results of amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Kappa test was used to analyze the consistency of the two methods.Results:The EGFR sensitive mutations were found in 31 cases (49.2%) by ddPCR in peripheral blood of 63 patients with NSCLC. Among them, 1 case (1.6%) had G719X, 12 cases (19.0%) had E19-Del, 11 cases had T790M (17.5%), 7 cases (11.1%) had L858R. Seven cases (22.6%) of the patients had double mutations. In comparison, the above 4 mutations were found in 26 cases (41.3%) by the ARMS-PCR method, with 0 case, 12 cases (19.0%), 6 cases (9.5%), and 8 cases (12.7%), respectively, including 5 cases (19.2%) with double mutations. L858R in one case was positive when detected by ARMS-PCR, while it was negative when detected by ddPCR. The consistency rate of the two methods was 90.3% (κ = 0.8, P < 0.05). The median abundance of EGFR mutations in peripheral blood ctDNA of 31 cases was 1.7% (range 0.04%-23.60%). The median abundance of E19-Del was 2.50% (0.35%-22.70%), that of T790M was 0.6% (0.04%-14.00%), and that of L858R was 2.3% (0.20%-23.60%). Ten cases with the abundance of EGFR mutations < 1% when detected by ddPCR, accounting for 32.6% (10/31) of total patients with mutations, but only 5 cases of them were detected by ARMS-PCR. The detection rate of T790M by ddPCR in patients who had received tyrosine kinase inhibitor (TKI) and had acquired drug resistance was 57.9% (11/19), while it was 0 in patients without TKI treatment. Among patients with T790M mutation, 1 case had a mutation abundance < 0.1%, 7 cases had a mutation abundance of 0.1%-2.0%, 3 cases had a mutation abundance > 2.0%.Conclusions:The ddPCR provides a non-invasive, highly sensitive and absolutely quantitative method for detecting EGFR mutations in peripheral blood ctDNA of NSCLC patients. It provides a new detection method for EGFR-TKI targeted therapy in NSCLC patients with difficult sampling or with acquired drug resistance who need to repeatedly sample. The approach provides an important basis for the individualized targeted therapy.

Objective:Nuclear magnetic resonance spectroscopy (NMR) was used to detect the species and content of metabolites in urine of patients with inherited metabolic diseases, and to explore the application value of NMR technology in the diagnosis of inherited metabolic diseases.Methods:Urine samples were collected from 20 patients with inherited metabolic diseases diagnosed in Xinhua Hospital, Shanghai Jiaotong University School of Medicine from March to June 2019, including 9 cases of methylmalonic acidemia (MMA). NMR pulse length-based concentration determination and Gas chromatography mass spectrometry (GC/MS) semi-quantitative method were used to detect the composition of metabolites in urine samples of patients with inherited metabolic diseases, and the levels of abnormal metabolites in the two methods were analyzed.Results:NMR technology can detect the levels of characteristic metabolites significantly increased in the urine of patients with MMA, isovalerinemia, glutaric acidemia, propionic acidemia, 3-methylcrotonyl-CoA carboxylase deficiency, ornithine carbamyltransferase deficiency, Citrin deficiency, Canavan disease, tyrosinemia and lysinuria protein intolerance. The average is 8 times of the upper limit of the reference value, and the highest is 545 times. Compared to GC/MS, NMR technology can detect the levels of various metabolites such as organic acids, amino acids and sugars. In 9 cases of untreated MMA,the median levels of methylmalonic acid and 3-hydroxypropionic acid in NMR [1 800 (180-12 000) and 50 (0-270) mmol/mol Cr] were higher than the reference values (0-31, 0-35). The median levels of methylmalonic acid and methylmalonic acid in GC/MS [136.56 (43.79-518.67) and 4.87 (1.52-7.52)] were higher than the reference values (0-4 and 0-0.7).Conclusions:NMR and GC/MS technologies are specific for the diagnosis of organic acidemia. The primary component detected by GC/MS is organic acid. NMR technology can break through this limitation and measure the level of various metabolites in urine, which provides a more theoretical basis for the diagnosis and research of inherited metabolic disease.

Objective:To analyze the disease spectrum of lysosomal storage disorders(LSDs) and explore the prevalent distributions of different LSD types in one center in Shanghai.Methods:A retrospective analysis was made.A total of 5 476 suspected LSD patients, including 3 415 males and 2 061 females, with a median age of 4 years(1 day to 72 years), were collected from Xinhua Hospital, Shanghai Jiaotong University School of Medicine from August 2008 to May 2022.The activity of different lysosomal enzymes was detected by fluorescent and biochemical methods.Results:A total of 1 520 patients were diagnosed with LSDs, including 972 males and 548 females, with a median age of 4 years(1 day to 59 years), involving 19 different subtypes.Mucopolysaccharidosis(MPS) was the most common type among LSDs, with a frequency of 45.46%(691/1 520), followed by sphingolipidoses [33.88%(515/1 520)] and glycogen storage disease type Ⅱ [16.05%(244/1 520)] successively.MPS Ⅱ was the most common type in MPS, with a frequency of 45.73%(316/691), followed by MPS ⅣA [22.87%(158/691)]. Niemann-Pick A/B, Gaucher, and Krabbe diseases were common in Sphingolipidoses patients, with frequencies of 37.09%(191/515), 34.37%(177/515), and 10.29%(53/515), respectively.Conclusions:LSDs are common genetic metabolic diseases, especially MPS and sphingolipidoses.Newborn screening for LSDs should be carried out timely so that the patients can be treated early and their prognosis can be improved.

Objective:To explore KRAS, NRAS, BRAF gene mutations and microsatellite instability(MSI) in colorectal cancer tissues as well as their correlation with the clinicopathological characteristics of patients.Methods:The clinicopathological data of 473 colorectal cancer patients in Shanxi Province Cancer Hospital from October 2020 to May 2021 were retrospectively analyzed. The mutation status of KRAS, NRAS and BRAF gene in the paraffin tissues were detected by using amplification refractory mutation system (ARMS) method. Polymerase chain reaction (PCR)-capillary electrophoresis was used to analyze MSI status, and the correlation of the clinicopathological characteristics of patients with gene mutations and MSI status was analyzed.Results:The mutation rates of KRAS, NRAS and BRAF were 45.03% (213/473), 2.96% (14/473) and 5.50% (26/473), respectively in 473 patients with colorectal cancer. No case harbored both 2 gene mutations was detected. The mutation rate of KRAS gene in well differentiated adenocarcinoma was higher than that in poorly differentiated adenocarcinoma [47.4% (175/369) vs. 36.5% (38/104), χ2 = 3.89, P = 0.049]. NRAS mutation rate in female was higher than that in male [5.0% (10/202) vs. 1.5% (4/271), χ2 = 4.86, P = 0.027], and the NRAS mutation rate in patients with tumor diameter ≤ 3 cm was higher than that in those with tumor diameter >3 cm [7.1% (7/98) vs. 1.9% (7/375), P = 0.013]. BRAF mutation rate of tumors located in colon was higher than that in rectum [11.7% (20/171) vs.2.0% (6/302), χ2 = 19.81, P < 0.001]; BRAF mutation rate in poorly differentiated tumor was higher than that in well differentiated tumor [10.6% (11/104) vs. 4.1% (15/369), χ2 = 6.62, P = 0.010]; BRAF mutation rate in patients with mucus was higher than that in those without mucus [10.9% (11/101) vs. 4.0% (15/372), χ2 = 7.19, P = 0.007]; BRAF mutation rate in patients with lymphatic metastasis was higher than that in patients without lymphatic metastasis [8.2% (15/182) vs.3.8% (11/291), χ2 = 4.29, P = 0.038]. The incidence of high frequency MSI (MSI-H) in 473 colorectal cancer tissues was 7.19% (34/473). The incidence of MSI-H in colon was higher than that in rectum [14.0% (24/171) vs. 3.3% (10/302), χ2 = 18.82, P < 0.001]; the incidence of MSI-H in patient with poor differentiated tumor was higher than that in those with well differentiated tumor [17.3% (18/104) vs. 4.3% (16/369), χ2 = 20.46, P < 0.001]; the incidence of MSI-H in patients with mucus was higher than that in those without mucus [11.9% (12/101) vs. 5.9% (22/372), χ2 = 4.24, P = 0.039]; and the incidence of MSI-H in patients without lymphatic metastasis was higher than that in patients with lymphatic metastasis [10.0% (29/291) vs. 2.7% (5/182), χ2 = 8.75, P = 0.003]. In addition, the incidence of MSI-H was on the rise in patients with BRAF mutation ( P < 0.001). Conclusions:KRAS, NRAS, BRAF gene mutations and MSI status are correlated with the clinicopathological characteristics of patients with colorectal cancer; there is a close relationship between MSI-H and BRAF mutation.

OBJECTIVE3-Methylcrotonyl-coenzyme A carboxylase deficiency (MCCD) is an autosomal recessive inborn error of leucine catabolism. The cases suspected as MCCD detected by neonatal screening are not rare. The aim of the study was to investigate the clinical outcomes in cases suspected as MCCD by neonatal screening. The second aim was to investigate the mutation spectrum of MCC gene in Chinese population and hotspot mutation.

METHODForty-two cases (male 33, female 9) , who had higher blood 3-hydroxy-isovalerylcarnitine (C5-OH) levels(cut-off <0.6 µmol/L) detected by neonatal screening using MS/MS, were recruited to this study during Sept.2011 to Mar.2013. The C5-OH concentrations were [0.84 (0.61-20.15) µmol/L] in 42 cases at the screening recall. Five cases were firstly diagnosed as maternal MCCD, 6 cases as benign MCCD and 31 cases were suspected as MCCD. To follow up the height, weight, mental development, blood C5-OH concentrations and urinary 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxy isovalerate (3-HIVA) in order to investigate the clinical outcome. The MCCC1 and MCCC2 gene mutation were analyzed for some cases. The novel gene variants were evaluated, and the influence of novel missense variants on the protein structure and function were predicted by PolyPhen-2, SIFT, UniProt and PDB software.

RESULT(1) Forty-two cases had no symptoms, their physical and mental development were normal in the last visit at the median ages of 29 months, the oldest age of follow up was nearly 9 years. (2) Gene mutation analysis was performed for 29 cases with informed consent signed by parents.Fourteen different mutations were identified in 19 cases. The mutations in MCCC1 gene accounted for 86%, the most common mutation was c.ins1680A, (accounted for 40%). Nine kinds of novel variant were detected including 211AG>CC/p.Q74P, c.295G>A/p.G99S, c.764A>C/p.H255P, c.964G>A/p. E322K, c.1331G>A/p.R444H, c.1124delT, c.39_58del20, c.1518delG, c.639+2T>A.Other 3 kinds of mutation in MCCC1 gene and 2 kinds of mutation in MCCC2 gene have been reported previously; the amino acid of mutant positions of five kinds of novel missense variant are almost highly conserved. These missense variants were predicted to cause change of human MCC protein side chain structure by changing hydrogen bonding, size of amino acid residue and electric charge, and predicted to damage the protein function possibly according to PolyPhen-2 and PDB analysis. So these novel variants may be disease-causing mutations. No mutation were detected in 10 cases. (3) Blood concentrations of C5-OH when screening, recall and end of follow-up in maternal MCCD was 3.50 (1.63-11.43), 1.84 (1.00-9.30), 0.27 (0.26-5.81) µmol/L. There was a significant downward trend.In contrast, benign MCCD group was 8.20 (3.60-9.60), 9.67 (3.88-20.15), 23.0 (5.87-49.10) µmol/L.It showed a rising trend. Children's urinary 3-MCG of benign MCCD group was found abnormally elevated in 4 cases (100%) when they were recalled.

CONCLUSIONA certain number of cases with MCCD or suspected as MCCD in this study had no symptoms and normal physical and mental development after follow-up to oldest age of nearly 9 years. The mutation in MCCC1 gene is common, nine novel mutations were found, c.ins1680A may be a hotspot mutation in Chinese population. The urinary GC/MS analysis and blood MS/MS analysis for mother should be routinely performed for all cases with high blood C5-OH level detected by neonatal screening.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Carbon-Carbon Ligases ; blood ; deficiency ; genetics ; Carnitine ; analogs & derivatives ; blood ; Child ; Child, Preschool ; DNA Mutational Analysis ; Female ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Neonatal Screening ; Tandem Mass Spectrometry ; Urea Cycle Disorders, Inborn ; blood ; diagnosis ; enzymology ; genetics