Adrenal Hyperplasia, Congenital ; complications ; metabolism ; Body Height ; Growth Disorders ; etiology ; Humans ; Risk Factors ; Steroid 21-Hydroxylase ; metabolism

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Abstract: Objective　To analyze the serotype distribution, drug resistance rate and drug resistance gene carrying of Streptococcus pneumoniae isolates in hospitalized patients, and evaluate the coverage of the vaccine to the serotype of Streptococcus pneumoniae in this area, so as to provide reference for the rational use of antibiotics in clinic. Methods　A total of 150 strains of non-repetitive Streptococcus pneumoniae isolated from inpatients from January 2015 to December 2019 were collected for serotyping and antimicrobial sensitivity test. The carrying rates of pbp2b, ermB and tetM were detected by PCR. Results　The PCR classification rate of 150 strains of Streptococcus pneumoniae was 93.1%, and the classification rate of capsular swelling test was 100%, and a total of 19 serotypes were divided, mainly 19F and 6B. Children's serotypes were predominantly 19F, 6B, and 15A; adult serotypes were predominantly 19F, 14, and 23F. The coverage rates of the PCV7, PCV10, PCV13 and PPV23 vaccines were 36.8%, 42.1%, 57.9% and 68.4%, respectively. Strains with serotypes of 19F, 6B, 3, and 23F had higher rates of resistance to antimicrobials. The sensitivity of Streptococcus pneumoniae to penicillin was greater than 96.0%. Antimicrobials with significant differences in resistance rates between invasive and non-invasive strains were penicillin, moxifloxacin, and levofloxacin. The percentage of strains carrying both ermB and tetM resistance genes was 96.0%, and the concordance rate between pbp2b, ermB and tetM resistance genes and the resistance phenotype was >98.0%. A total of 10 multi-resistance combinations were detected, with a multi-resistance rate of 62.6%, and the multi-drug resistance pattern of Streptococcus pneumoniae was mainly concentrated in the 19F and 6B serotypes. Conclusion　There are significant age differences in the serotypes of Streptococcus pneumoniae in this area. The vaccine currently used has low coverage in this region and therefore offer limited protection to the population. The drug resistance rates of Streptococcus pneumoniae varied significantly among serotypes. Erythromycin and tetracycline are not recommended for clinical treatment of Streptococcus pneumoniae. Penicillin can still be used as the first choice for clinical treatment of Streptococcus pneumoniae infection.

OBJECTIVETo report the results of clinical characteristics, enzyme activity determination and mutation analysis of GLB1 gene in a Chinese patient with mucopolysaccharidosis (MPS) type IVB (Morquio B disease).

METHODA 14-year-old Chinese boy with MPS type IVB was firstly diagnosed by blood leucocytes galactosamine-6-sulfate sulfatase (GALNS) and β-galactosidase (GLB1) determination, who was characterized by short stature, multiplex skeletal abnormalities, difficulty in walking. PCR-sequencing analysis was applied to detect the mutations in GLB1 of the patient.

RESULTThe patient was characterized by dwarfism, pectus carinatum, kyphosis, normal intelligence, and no neurologic damage of spasms, linguistic capacity and so on. The patient had normal GALNS enzyme activity and very low GLB1 enzyme activity [5.03 nmol/(h·mg) vs. normal value 118 - 413 nmol/(h·mg) ] in leukocytes. A compound heterozygous missense mutations c.442C > T(p.R148C)/c.1454A > G(p.Y485C) in GLB1 gene were detected in this patient. The mutation p.Y485C is a novel variant. With the method of gene analysis of new variant, the mutation p.Y485C was considered to be a pathogenic mutation.

CONCLUSIONThe MPS IVB patient showed severe multiple skeletal deformities, normal intelligence, no neurologic damage and very low GLB1 enzyme activity, who carries compound heterozygous mutations p.R148C/p.Y485C. The mutation p.Y485C in GLB1 gene may be a novel pathologic mutation of MPS type IVB.

Adolescent ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Chondroitinsulfatases ; genetics ; metabolism ; DNA Mutational Analysis ; Humans ; Joints ; pathology ; Male ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; enzymology ; genetics ; pathology ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; Radiography ; Spine ; diagnostic imaging ; pathology ; beta-Galactosidase ; genetics ; metabolism

OBJECTIVETo observe the effects of puerarin on ADRP gene mRNA expression in fatty tissue of type 2 diabetes mellitus rats (T2DM).

METHODWiastar rats of T2DM model were made by feeding with high glucose and fat diet and injecting with small dose of streptozocin (25 mg x kg(-1)). 40 model rats were randomly divided into model control group and three puerarin groups (40, 80, 160 mg x kg(-1)), another 10 rats were selected as normal control group. FBG and FINS were measured to calculate IR after rats were injected consecutively for 6 weeks. The level of ADRP gene mRNA in fatty tissue was determined by RT-PCR after rats were injected eight weeks.

RESULTCompared with model control group, high and middle dosage of puerarin can decreased ADRP gene mRNA expression in fatty tissue obviously, FBG, IR level in each puerarin group and FINS in high and middle dosage puerarin groups decreased obviously.

CONCLUSIONPuerarin can decrease the blood glucose level of T2DM by downregulating ADRP mRNA expression and depressing the insulin resistance.

Adipose Tissue ; drug effects ; metabolism ; Animals ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; drug therapy ; genetics ; Female ; Gene Expression Regulation ; drug effects ; In Vitro Techniques ; Isoflavones ; pharmacology ; Male ; Membrane Proteins ; genetics ; Perilipin-2 ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Vasodilator Agents ; pharmacology

OBJECTIVEChitotriosidase (CT) is a plasma biomarker for Gaucher disease (GD), the enzyme activity is usually markedly elevated in plasma of Gaucher patients, and it was reported that levels of plasma chitotriosidase activity was mildly-moderately increased in patients with Niemann-Pick disease (NPD). The aim of this study was to compare chitotriosidase activity using 4-methylumbelliferyl-β-D-N, N', N″-triacetyl-chitotrioside (4MU-C3) with 4-methylumbelliferyl 4-deoxy-β-D-chitobiose (4MU-4dC2) as substrates, and apply chitotriosidase activity measurement to help clinical determination of GD and NPD, and to monitor therapy in GD patients.

METHODPlasma of 45 healthy individuals, 31 patients with GD and 9 patients with NPD type A/B was collected from outpatient clinics of the Department of Pediatric Endocrinologic, Genetic and Metabolic Diseases, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Plasma chitotriosidase activity was measured with the substrates 4MU-C3 and 4MU-4dC2 respectively. Determinations were based on the methods described by Hollak et al and Rodrigues et al. Meanwhile, common mutation dup24 of the human chitotriosidase gene was detected.

RESULT(1) Chitotriosidase activity when measured with 4MU-4dC2 gave higher values than 4MU-C3. In the healthy controls chitotriosidase activity was increased 3.7-fold when the 4MU-dC2 was used as substrate as compared with the 4MU-C3 (Z = -4.703, P < 0.001). In the untreated GD patients, the median value was increased 794-fold and 610-fold of the control subjects (Z = -3.823, P < 0.001) when the enzyme was measured with two substrates respectively. In the GD patients during therapy, chitotriosidase activity was increased 134-fold and 79-fold, and after changing therapeutic dose chitotriosidase activity was increased 215-fold and 118-fold of the controls (Z = -2.521, P < 0.05). In the NPD patients chitotriosidase activity was increased 8-fold and 14-fold of the controls (Z = -1.604, P = 0.109). (2) Consistent with the results of chitotriosidase activity, 30 of 85 (35.3%) individuals were homozygotes of dup24 mutation, which are completely chitotriosidase enzyme deficiency. Among GD patients with wild-type and heterozygotes for the dup24 mutation, chitotriosidase activity highly increased in the plasma compared with the controls.

CONCLUSIONThe use of 4MU-4dC2 as substrate makes chitotriosidase activity measurement more sensitive. The determination of plasma chitotriosidase activity is a useful tool to assist the clinical identification of Gaucher disease, and to monitor enzyme replacement therapy (ERT) of non-chitotriosidase deficient GD patients. Chitotriosidase activity determination has no value in the clinical identification of NPD.

Adolescent ; Adult ; Blood Chemical Analysis ; methods ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Gaucher Disease ; blood ; enzymology ; genetics ; Genotype ; Heterozygote ; Hexosaminidases ; blood ; genetics ; metabolism ; Humans ; Infant ; Male ; Middle Aged ; Mutation ; Niemann-Pick Diseases ; blood ; enzymology ; genetics ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Young Adult

OBJECTIVECongenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive inherited disorder, characterized by deficiency of adrenal and gonadal steroid hormones. Recent studies have shown that mutations in the gene for steroidogenic acute regulatory protein (StAR) cause this most severe genetic disorder in steroid hormone biosynthesis. StAR is a mitochondrial protein promotes cholesterol transfer from outer mitochondrial membrane to the inner mitochondrial membrane, where the cholesterol serves as a substrate for P450scc and initiates steroidogenesis. So far, more than 30 different mutations in the StAR gene have been found in the patients with CLAH from various ethnic groups. None of CLAH patients in the Chinese population has been previously reported. In the present study we analyzed the StAR gene in a Chinese patient with CLAH.

METHODSThe patient who was a 19-yr-old phenotypic female, has a 46, XY karyotype. Endocrinological evaluation was performed. Genomic DNA samples were abstracted from the bloods of the patient and his parents. Polymerase chain reaction (PCR), direct DNA sequencing, family analysis and restriction enzyme digestion analysis were used to detect and confirm the mutations of StAR gene.

RESULTSEndocrine evaluation of the patient showed extremely elevated basal concentrations of serum ACTH and gonadotropin and minimal concentration of gonadal steroids. An ACTH stimulation test indicated basal serum dehydroepiandrosterone and 17-hydroxyprogesterone were lower than normal detectable range and had no obvious increase after the ACTH stimulation. Automatic sequencing of 7 exons of the StAR gene with the polymerase chain reaction products of the genomic DNA revealed compound heterozygous for a novel nonsense mutation Q77X in exon 3 and the frameshift mutation 838delA in exon 6. The father carried Q77X mutation and the mother carried 838delA mutation. The restriction enzyme site of the Q77X mutation was examined by endonucleotidase BfaI. Furthermore, this mutation was not found in a series of 20 alleles of normal individuals.

CONCLUSIONQ77X is the novel mutation found in the patient with CLAH. Q77X and 838delA compound mutations could inactivate the StAR function and give rise to clinically manifest CLAH. This case is the first Chinese patient with CLAH identified by molecular genetic analysis. DNA-based analysis of StAR gene will be helpful for the diagnosis of CLAH.

Adrenal Hyperplasia, Congenital ; complications ; genetics ; Adrenal Insufficiency ; etiology ; Female ; Genotype ; Gonadal Steroid Hormones ; deficiency ; Humans ; Mutation ; Phenotype ; Phosphoproteins ; genetics ; Young Adult

OBJECTIVEThe new technology of tandem mass spectrometry is exerting a significant impact on the diagnostics of inborn metabolic errors, and allows to detect a number of these disorders in a single step. The aim of the present study was to establish a dry blood filter paper method for amino acid and acylcarnitine profiles test using tandem mass spectrometry and to apply the method for selective screening in high risk children with inborn error of metabolism.

METHODThe study group consisted of 104 high risk cases of inborn error of metabolism from 5 pediatric hospitals in Shanghai and Beijing were tested from November 2002 to June 2003; 77 were males and 27 females, the means age was 4.8 +/- 4.2 years. These patients had mental retardation, slow development, psychological abnormalities, muscle hypotonia, jaundice, hepatosplenomegaly, recurrent vomiting, and convulsion. Laboratory examinations suggested metabolic acidosis, hypoglycemia, hyperammonemia and hyperlactacidemia. Phenylketonuria was excluded in this study by routine phenylalanine screening. The control group consisted of 308 children, 170 males and 138 females. The blood was collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCl. After preparation, the samples were analyzed by tandem mass spectrometry (API 2000).

RESULTSTen of 104 patients (9.6%) were positive in our selective screening program, including one with tyrosinemia, one with homocystinuria, one with hyperornithinemia, two with methylmalonic acidemia, one with propionic acidemia, one with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, two with medium chain acyl-CoA dehydrogenase deficiency, and one with carnitine palmitoyl transferase type II deficiency.

CONCLUSIONThe authors established a fast, accurate and sensitive tandem mass spectrometry method for amino acid and acylcarnitine profiles analysis, nearly 30 metabolic diseases including amino acid disorders, organic acid disorders and fatty acid oxidation disorders could be detected, most of the diseases that cause death and disability represent preventable entities by early diagnosis and treatment. The results indicated the importance of selective screening for high risk patients with inborn error of metabolism.

Adolescent ; Amino Acids ; blood ; Carnitine ; analogs & derivatives ; blood ; Child ; Child, Preschool ; China ; Female ; Humans ; Infant ; Male ; Mass Screening ; Mass Spectrometry ; methods ; Metabolism, Inborn Errors ; blood ; diagnosis ; Pilot Projects ; Risk Factors

OBJECTIVEMany children were found to have low free carnitine level in blood by tandem mass spectrometry technology. In some of the cases the problems occurred secondary to malnutrition, organic acidemia and other fatty acid oxidation metabolic diseases, and some of cases had primary carnitine deficiency (PCD). In the present article, we discuss the diagnosis of PCD and evaluate the efficacy of carnitine in the treatment of PCD.

METHODWe measured the free carnitine (C0) and acylcarnitine levels in the blood of 270 000 neonates from newborns screening program and 12 000 children with suspected clinical inherited metabolic diseases by tandem mass spectrometry. The mutations of carnitine transporter protein were tested to the children with low C0 level and the diagnosis was made. The children with PCD were treated with 100 - 300 mg/kg of carnitine.

RESULTSeventeen children were diagnosed with PCD, 6 from newborn screening program and 11 from clinical patients. Mutations were found in all of them. The average C0 level [(2.9 ± 2.0) µmol/L] in patients was lower than the reference value (10 µmol/L), along with decreased level of different acylcarnitines. The clinical manifestations were diverse. For the 6 patients from newborn screening, 4 were asymptomatic, 1 showed hypoglycaemia and 1 showed movement intolerance from 2 years of age. For the 11 clinical patients, 8 showed hepatomegaly, 7 showed myasthenia, 6 showed cardiomyopathy, 1 showed chronic abdominal pain, and 1 showed restlessness and learning difficulty. Among these patients, 14 cases were treated with carnitine. Their clinical symptoms disappeared 1 to 3 months later. The C0 level in the blood rose to normal, with the average from (4.0 ± 2.7) µmol/L to (20.6 ± 8.3) µmol/L (P < 0.01). However, the level was still lower than the average level of healthy children [(27.1 ± 4.5) µmol/L, P < 0.01].

CONCLUSIONSeventeen patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry, and gene mutation test. Large dose of carnitine had a good effect in treatment of the PCD patients.

Cardiomyopathies ; diagnosis ; drug therapy ; genetics ; Carnitine ; analogs & derivatives ; blood ; deficiency ; genetics ; Child, Preschool ; DNA Mutational Analysis ; Female ; Follow-Up Studies ; Humans ; Hyperammonemia ; diagnosis ; drug therapy ; genetics ; Infant ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis ; drug therapy ; genetics ; Mutation ; Neonatal Screening ; methods ; Organic Cation Transport Proteins ; deficiency ; genetics ; Reference Values ; Tandem Mass Spectrometry

OBJECTIVEWith the emergence of enzyme replacement therapy and progress in bone marrow transplantation, treatment of mucopolysaccharidosis (MPS) is much more promising than ever. In order to benefit from these therapies, determination of the defective enzyme is the prerequisite for any individual patient. To make definite diagnosis for patients suspected of having MPS clinically, the authors established six lysosomal enzymatic assays for leucocytes, including alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine 6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, which are the corresponding enzymes of type I, type II, type IVA, type IVB, type VI, and type VII, respectively.

METHODSeventy patients suspected of having MPS were enrolled from outpatient clinics of the Department of Pediatric Endocrinologic, Genetic and Metabolic Diseases in Xinhua Hospital. Their ages spanned from 10 months to 25 years with the average age 5.7 years. Of them 49 were male and 21 were female. Leukocytes were isolated with Dextran from peripheral blood of suspected patients. Activity of leukocyte alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine 6-sulfatase, beta-galactosidase, beta-glucuronidase were measured using their specific artificial fluorescent substrates, while arylsulfatase B were determined by colorimetric assay with dipotassium 2-hydroxy-5-nitrophenyl sulfate as the substrate.

RESULTOf the 70 clinically suspected cases totally 47 were confirmed of having mucopolysaccharidosis, of whom 7 cases were type I, 28 cases type II, 12 cases type IVA. These data show that type II is the predominant form of MPS in China, succeeded by MPS type IVA. It was also noted that type II has the most variable clinical manifestations and 8 out of 12 type IVA patients had the unique lax joints.

CONCLUSIONThe present study suggest that type II might be the predominant form of MPS cases in China, followed by type IVA and type I.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Humans ; Iduronate Sulfatase ; metabolism ; Infant ; Infant, Newborn ; Male ; Mucopolysaccharidoses ; classification ; diagnosis ; enzymology ; Young Adult