Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Clinical characteristics of 111 cases with mucopolysaccharidosis ⅣA / 中华儿科杂志

Objective: To analyze the clinical characteristics of patients with Mucopolysaccharidosis ⅣA (MPS ⅣA). Methods: A retrospective study was conducted on 111 patients with MPS ⅣA in Xinhua Hospital of Shanghai Jiao Tong University School of Medcine from December 2008 to August 2020, confirmed by enzyme activity and genetic testing. General situation, clinical manifestations and enzyme activity test results were analyzed. According to the clinical manifestations, it can be divided into severe, intermediate and mild group. The independent sample t test was used to compare the birth body length and weight of children with that of normal boys and girls, and group comparisons of enzyme activities were evaluated by median test. Results: One hundred and eleven unrelated patients, 69 males and 42 females, were classified into 3 subtypes: severe (n=85), intermediate (n=14), and mild (n=12). The age at symptom onset were 1.6 (1.0, 3.0) years, and at diagnosis were 4.3 (2.8, 7.8) years. Skeletal manifestations were observed in all patients and consisted mainly of pectus carinatum (96/111, 86.5%), motor dysfunction (78/111, 70.3%), spinal deformity (71/111, 64.0%), growth retardation (64/111, 57.7%), joint laxity (63/111, 56.8%) and genu valgum (62/111, 55.9%). Eighty-eight patients (88/111, 79.3%) with MPS ⅣA were also along with non-skeletal manifestations, mainly including snoring (38/111, 34.2%), coarse faces (34/111, 30.6%), and visual impairment (26/111, 23.4%). The most common skeletal manifestation was pectus carinatum (79 cases), and non-skeletal manifestation was snoring (30 cases) and coarse faces (30 cases) in severe patients, pectus carinatum (13 cases) and snoring (5 cases) in intermediate type, motor dysfunction (11 cases) and snoring (3 cases) and visual impairment (3 cases) in mild patients. The height and weight of severe patients began to fall below -2 s at 2-<5 years and 5-<7 years, respectively. At the age of 10-<15 years, the standard deviation score of the height of severe patients reached (-6.2±1.6) s in males and (-6.4±1.2) s in females, and the score of weight got (-3.0±1.1) s in males and (-3.5±0.5) s in females. The height of intermediate patients began to fall below -2 s at the age of 7-<10 years, and the standard deviation score of height were -4.6 s and -3.6 s in 2 males, and -4.6 s and -3.8 s in 2 females at the age of 10-<15 years. The weight remained within -2 s in 72.0% (18/25) of intermediate patients compared to age-matched healthy children. In the mild patients with MPS ⅣA, the mean standard deviation score of height and weight was within -2 s. The enzyme activities of mild patients (2.02 (1.05, 8.20) nmol/(17 h·mg)) were both significantly higher than that of intermediate (0.57 (0.47, 0.94) nmol/(17 h·mg)) and severe (0.22 (0, 0.59) nmol/(17 h·mg)) patients (Z=9.91, 13.98, P=0.005, 0.001), and the enzyme activity of intermediate patients was significantly higher than that of severe patients (Z=8.56, P=0.010). Conclusions: The clinical manifestations of MPS ⅣA are charactered by pectus carinatum, motor function impairment, spinal deformity and growth retardation. The clinical characteristics, growth rate and enzyme activity differ among the 3 subtypes of MPS ⅣA.

Radioanatomic study on the role of Hadad-Bassagasteguy flap in skull base reconstruction in endoscopic endonasal approach / 中华耳鼻咽喉头颈外科杂志

Objective: To evaluate the value of Hadad-Bassagasteguy flap (HBF) in endoscopic endonasal approaches (EEA) skull base reconstruction by radioanatomic measurements on CT of the skull base of Chinese adults. The following data in terms of anterior skull base defect and reconstruction, sphenoid platform area and middle skull base defect and reconstruction including sphenoid platform and sella area, clivus area defect and reconstruction, and HBF were collected and assessed. Methods: CT image data of 42 Chinese adults were selected to obtain radioanatomic measurement data related to HBF, anterior skull base defect and reconstruction, middle skull base defect and reconstruction, and defect and reconstruction of clivus area. SPSS 26.0 software was used to analyze the data. Results: The radioanatomic measurement data about HBF and skull base of 42 Chinese adults were obtained. The width of the leading edge of HBF [(37.49±2.86) mm] was 6 mm more than the anterior skull base width at the level of the anterior ethmoidal artery [(30.87±8.61) mm], and the width of the trailing edge of HBF [(42.61±3.95) mm] was also 6 mm more than the anterior skull base width at the level of the sphenoethmoidal junction [(26.79±2.79) mm]. The total length of HBF including the pedicle [(79.68±4.96) mm] was 6 mm more than the length of the anterior skull base reconstruction [(54.06±8.67) mm], and the length of HBF without pedicle [(46.27±3.14)] mm was 6 mm more than the length of anterior skull base defect [(30.87±8.61) mm]. The trailing edge width was 6 mm more than the planum sphenoidal width at the level of the optic strut [(30.87±8.61) mm]. The total length of HBF including the pedicle was 6 mm more than the length of the planum sphenoidal, and the sella reconstruction [(64.44±10.25) mm], also was 6 mm more than the length of the planum sphenoidal reconstruction [(73.61±8.28) mm]. The length of HBF without pedicle was 6 mm more than the length of the planum sphenoidal, and the sella defect [(27.88±3.74) mm], also was 6 mm more than the length of the planum sphenoidal defect [(15.50±3.38) mm]. The width of the leading edge of HBF and the width of the trailing edge were both 6 mm more than the width of clivus reconstruction at the level of the foramen lacerum [(21.68±2.30) mm]. The total length of HBF including pedicles was 6 mm more than the clivus reconstruction length [(67.09±5.44) mm], while the length of HBF without pedicles was also 6 mm more than the clivus defect length [(37.19±3.80) mm]. Conclusions: In this study, the radiosanatomic measurements ensured that HBF could provide sufficient tissue flap for the reconstruction of the anterior skull base and sphenoid plateau and extend the reconstruction area to sella and clivus. Preoperative radiosanatomic measurement can be used to predict the size of HBF required for skull base reconstruction, which provides important guidance for flap harvest.

Quantitative Proteomics Using Isobaric Labeling: A Practical Guide / 基因组蛋白质组与生物信息学报·英文版

In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.

Research progress of circular RNA in ophthalmic diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#With the rapid development of molecular biology technologies such as whole-genome testing in the past decade, non-coding RNA has been deeply explored. Circular RNA(circRNA)gradually becomes a hot topic in recent years, which plays an important role in the occurrence and development of a variety of diseases, and relevant studies have been carried out in ophthalmological diseases. This article will review the mechanism, basic research, and clinical findings of circRNA in various ophthalmic diseases, and briefly discuss the potential clinical significance of circRNA in ophthalmic diseases.

Very-long chain acyl coenzyme A dehydrogenase deficiency / 中国实用儿科杂志

Very long chain acyl-CoA dehydrogenase deficiency(VLCADD)is a disorder involving the initial step of fatty acid beta-oxidation in the mitochondrial matrix. VLCADD can present at various ages,from the neonatal period to adulthood,with symptoms including hypoglycemia,rhabdomyolysis,skeletal muscle weakness and cardiomyopathy,and poses the greatest risk of complications during intercurrent illness or after prolonged fasting. Early diagnosis,treatment,and surveillance can reduce mortality. The most common diagnostic evaluation methods are plasma acylcarnitine profiles and ACADVL gene molecular testing. Functional testing,including white blood cell or fibroblast enzyme assay,is a useful diagnostic adjunct if molecular sequencing alone is insufficient to deter-mine the diagnosis or uncharacterized mutations are identified. Treatment emphasizes the avoidance of fasting and often includes a specialized diet that is high carbohydrate/low longchain fat which is supplemented by medium chain triglycerides(MCT).very-long chain acyl coenzyme A dehydrogenase

Paying attention to screening,diagnosis and treatment of fatty acid oxidation disorders / 中国实用儿科杂志

Fatty acid oxidation disorders(FAOD)include more than 10 kinds of diseases,they all belong to autosomal recessive diseases and are common inherited metabolic diseases. Onset age of the patients with FAOD are from newborn to adult. The clinical manifestations were nonspecific,mainly manifested as liver disease,cardiomyopathy and muscle diseases. Detection of free carnitine and acylcarnitines in blood by tandem mass spectrometry and detection of gene mutations are important methods for diagnosis of such diseases. Tandem mass screening for neonatal screening is helpful for early diagnosis and early treatment of FAOD. Primary carnitine deficiency and multiple acyl-CoA dehydrogenase deficiency can be treated by specific therapeutic drugs with good effect. There are no specific drugs for other diseases,which need symptomatic treatment.

PD1/PD-L1 Blockade therapy for cancer:influencing factors,predictive biomarkers and combined treatments / 国际药学研究杂志

Recently,programmed cell death 1/programmed cell death 1 ligand(PD-1/PD-L1)blockade has elicited clinical re-sponses and long-term remissions in a subset of patients with a broad spectrum of cancers. Unfortunately,a substantial number of pa-tients failed to respond to PD1/PD-L1 blockade. In this paper,we summarize our current understanding of the key factors accounting for the difference on treatment,and evaluate the dependability of several potential predictive biomarkers guiding PD1/PD-L1 blockade in cancer therapy. We also discuss a variety of combined treatments based on PD1/PD-L1 blockade.

Effect of visual perception training combined with total nutrition meal sequential therapy on myopic amblyopia in preschool children / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To observe the therapeutic effect of visual perception training combined with total nutrition meal sequential therapy in the treatment of myopic amblyopia.·METHODS:Totally 73 children ( 135 eyes ) with myopic amblyopia were divided into control group ( 36 cases, 67 eyes) and treatment group (37 cases, 68 eyes) according to random number table. The control group were treated with traditional spectaculars and grating covering combined with fine eyesight training;the treatment group were treated with visual perception training combined with total nutrient meal sequential therapy. The visual acuity, diopter and average diopter of two groups were compared before and after treatment at 3, 6mo and 1a. The curative effect of two groups of children was compared after 1a treatment. And the adverse reactions were recorded in two groups during the treatment period. The recurrence rate of amblyopia in 1a follow-up was compared between two groups.·RESULTS: The difference of visual acuity between two groups was not significant at 3mo (P>0. 05). The visual acuity of the treatment group was significantly higher than that of the control group at 6mo and 1a (P<0. 05). There was no significant difference in diopter between the two groups after 3, 6mo and 1a (P>0. 05), but the average annual refractive changes in the treatment group were significantly lower than that in the control group ( P<0. 05). The basic cure rate and total effective rate of the treatment group were significantly higher than that of the control group (P<0. 05). There were no severe adverse reactions occurred between two groups during the treatment period. The recurrence rate of amblyopia in the treatment group was significantly lower than that in the control group (P<0. 05) after 1a follow-up.· CONCLUSION: Visual perception training combined with total nutrition meal sequential therapy in the treatment of myopic amblyopia in preschool children can significantly improve patients' visual acuity, reduce the average annual diopter changes, improve the therapeutic effect, reduce the recurrence rate of amblyopia.

Outcomes of patients with combined methylmalonic acidemia and homocystinuria after treatment / 中华儿科杂志

<p><b>OBJECTIVE</b>Combined methylmalonic acidemia with homocystinuria is a common form of methylmalonic acidemia in China. Patients with this disease can progress to death without timely and effective treatment. This study aimed to analyze the treatment outcomes of patients with combined methylmalonic acidemia and homocystinuria.</p><p><b>METHOD</b>From September 2004 to April 2012, 58 patients with combined methylmalonic acidemia and homocystinuria (34 males and 24 females) were diagnosed and treated in our hospital. Fifty cases were from clinical patients including 42 early-onset cases and 8 late-onset cases. Their age when they were diagnosed ranged from 18 days to 30.8 years. The other 8 cases were from newborn screening. All the patients were treated with vitamin B12, betaine, folic acid, vitamin B6, and L-carnitine. The physical and neuropsychological development, general laboratory tests, the levels of amino acids, acylcarnitines, and homocysteine in blood, and organic acids in urine were followed up.</p><p><b>RESULT</b>The follow-up period ranged from 1 month to 7.1 years. Three cases died (all were early-onset cases). In the other patients after treatment, the symptoms such as recurrent vomiting, seizures, lethargy, and poor feeding disappeared, muscle strength and muscle tension were improved, and general biochemical abnormalities such as anemia and metabolic acidosis were corrected. Among the surviving 55 cases, 49 had neurological impairments such as developmental delay and mental retardation. The median levels of blood propionylcarnitine and its ratio with acetylcarnitine, serum homocysteine, and urine methylmalonic acid were significantly decreased (P < 0.01), from 7.73 µmol/L (ranged from 1.5 to 18.61 µmol/L), 0.74 (ranged from 0.29 to 2.06), 97.3 µmol/L (ranged from 25.1 to 250 µmol/L) and 168.55 (ranged from 3.66 to 1032.82) before treatment to 2.74 µmol/L (ranged from 0.47 to 12.09 µmol/L), 0.16 (ranged from 0.03 to 0.62), 43.8 µmol/L (ranged from 17 to 97.8 µmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively.</p><p><b>CONCLUSION</b>Patients with combined methylmalonic acidemia and homocystinuria respond to a combined treatment consisting of supplementation of hydroxycobalamin, betaine, folic acid, vitamin B6 and L-carnitine with clinical and biochemical improvement. But the long-term outcomes are unsatisfactory, with neurological sequelae in most patients.</p>

Analysis of GALNS gene mutation in thirty-eight Chinese patients with mucopolysaccharidosis type IVA / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis (MPS) type IVA (MPS IVA) is an autosomal recessive lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) needed to degrade glycosaminoglycanes (GAGs), accumulation of GAGs in the tissue resulting in disorder of function. So far, the small number of articles about clinical study of Chinese MPS IVA were published and only one paper about gene mutation analysis was published. This study aimed to investigate the mutation spectrum and characteristic of GALNS gene in Chinese patients with MPS IVA who were diagnosed in our hospital.</p><p><b>METHOD</b>Thirty-eight patients from 36 families (male 17, female 21) were diagnosed as MPS IVA by GALNS activity determination [(0.85 ± 1.33) nmol/(17 h·mg)] and clinical symptoms during 2006-2012. The average age of diagnosis was (5.7 ± 3.6) years. Mutation analysis of GALNS gene performed performed by PCR-direct DNA sequencing for 38 patients. PCR-restriction fragment length polymorphism analysis was used for validating novel mutation, and also to assess amino acid conservation for novel missense variants in five different species. PolyPhen-2 tool was used to predict the possible impact of missense mutations on the structure and function of the human GALNS protein, etc. Analysis of GALNS activity and gene mutation in amniotic fluid were performed to provide the prenatal diagnosis for some families with MPS type IVA.</p><p><b>RESULT</b>(1) Thirty-eight kinds of mutation in GALNS gene were identified in 38 patients of them, 71% were missense mutations. p. M318R was a hot-spot mutation (21%) tested. Five kinds of mutation i.e., p. P163H, p.G168L, p. A324E, p. L366P and p. F452L were only found in Chinese patients with MPS IVA. Eighteen kinds of novel mutation were detected including p. E315K, p.G304D, p.R251Q, p.Y240C, p.G161E, p.N32D, p.L390P, p. D60E, p. P420S, W403C/T404S, p.L454P, for p.W405X, p. M1I, c.409_ c.420del12, c.1176_1178del3, c.1046delG, c.1188delG and IVS9-2A>C. (2) The polymorphism of novel missense variants were ruled out by the PCR-restriction fragment length polymorphism analysis and no related mutations were found in 50 normal controls. A splice site mutation IVS9-2A>C had been validated by reverse transcription PCR direct sequencing. The amino acid of mutant position of 10 kinds of missense variants are highly conserved and only p. L454 is moderately conserved position. These missense variants were predicted to cause damage to the structure and function of human GALNS protein possibly according to the PolyPhen-2 tool, so these novel missense variants may be disease-causing mutations. (3) Prenatal diagnosis was provided for 7 families and three fetuses were diagnosed as MPS IVA.</p><p><b>CONCLUSION</b>The GALNS gene mutation spectrum in Chinese patients with MPS IVA is really different from that in other countries, five kinds of mutation were only found in Chinese patients with MPS IVA. The reports of hot-spot mutation in Chinese patients were also different, and should be analyzed by more data of gene mutation analysis and epidemiological study.</p>

Clinical and mutational features of maternal 3-methylcrotonyl coenzyme deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report on 5 patients with maternal 3-methylcrotonyl coenzyme A carboxylase deficiency (MCCD) and to confirm the clinical diagnosis through mutation analysis.</p><p><b>METHODS</b>Five neonates with higher blood 3-hydroxy isovalerylcarnitine (C5-OH) concentration detected upon newborn screening with tandem mass spectrometry and their mothers were recruited. Urinary organic acids were analyzed with gas chromatography mass spectrometry. Gene mutation and protein function analysis were performed by PCR direct sequencing and PolyPhen-2 software.</p><p><b>RESULTS</b>Higher blood C5-OH concentrations (5.11-21.77 μmol/L) and abnormal 3-hydroxy isovalerate and 3-methylcrotonyl glycine in urine were detected in the five asymptomatic mothers, who were diagnosed as benign MCCD. Higher C5-OH concentration was also detected in their neonates by tandem mass spectrometry, which had gradually decreased to normal levels in three neonates. Four new variations, i.e., c.ins1680A(25%), c.203C > T (p.A68V), c.572T > C (p.L191P) and c.639+5G > T were detected in the MCCC1 gene, in addition with 2 mutations [c.1406G > T (p.R469L, novel variation) and c.592C > T (p.Q198X)]. The novel variations were predicted to have affected protein structure and function.</p><p><b>CONCLUSION</b>For neonates with higher C5-OH concentration detected upon neonatal screening, their mothers should be also tested to rule out MCCD. Mutations in MCCC1 gene are quite common.</p>

Diagnosis and treatment of isolated methylmalonic acidemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the clinical feature, therapeutic effect and prognosis of isolated methylmalonic acidemia.</p><p><b>METHODS</b>The clinical characteristics, laboratory findings, treatment and outcome of 40 patients were retrospectively analyzed. The main treatment was a low-protein diet supplemented with L-carnitine and special milk free of leucine, valine, threonine and methionine. Vitamin B12 was also given to cobalamin responders. The patients were followed up every 1-3 months.</p><p><b>RESULTS</b>Mutations in the MUT gene were identified in 30 of 33 patients who had accepted DNA testing. Thirty cases were treated and followed up regularly for from 1 month to 8 years. Eight cases had died, 8 had developed normal intelligence, among whom 4 from newborn screening were asymptomatic. Psychomotor developmental delay and mental retardation were present in 14 cases. The propionylcarnitine level, ratio of propionylcarnitine/acetylcarnitine in blood, methylmalonic acid and methylcitric acid levels in urine have decreased significantly, with the median values reduced respectively from 24.15 (7.92-81.02) μmol/L, 1.08 (0.38-6.01), 705.34 (113.79-3078.60) and 7.71 (0.52-128.21) to 10.50 (3.00-30.92) μmol/L, 0.63 (0.25-2.89), 166.23 (22.40-3322.21) and 3.96 (0.94-119.13) (P < 0.05).</p><p><b>CONCLUSION</b>The prognosis of isolated methylmalonic acidemia may be predicted with the enzymatic subgroup, age at onset and cobalamin responsiveness. Outcome is unfavorable in neonatal patients and those who were non-responsive to cobalamin.</p>

Analysis of clinical data and genetic mutations in three Chinese patients with tyrosinemia type I / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze clinical data and gene mutations in 3 Chinese patients with tyrosinemia type I, and to explore the correlation between genotypes and phenotypes.</p><p><b>METHODS</b>Three patients suspected with tyrosinemia I were tested by tandem mass spectrometry for the level of tyrosine, phenylalanine and succinylacetone in the blood, and by gas chromatography-mass spectrometry to determine the level of succinylacetone and organic acid in their urine. With the diagnosis established, the FAH gene was analyzed with polymerase chain reaction (PCR) and direct sequencing.</p><p><b>RESULTS</b>Two patients had acute onset of the disease, while another had subacute onset of the disease, with features including hepatomegaly and remarkably increased tyrosine and succinylacetone in the blood. Five mutations were detected in the FAH gene, which included c.455G>A (W152X), c.520C>T (R174X), c.974_976delCGAinsGC, c.1027 G>A (G343R) and c.1100 G>A (W367X), among which c.455G>A (W152X), c.974_976delCGAinsGC and c.1100 G>A (W367X) were not reported previously.</p><p><b>CONCLUSION</b>Tyrosinemia type I may be effectively diagnosed with the level of tyrosine and succinylacetone by tandem mass spectrometry and succinylacetone in the urine by gas chromatography mass spectrometry. Detection of underlying mutations mutations will be helpful for genetic counseling and further research.</p>

GLB1 gene mutation and clinical characteristics of a patient with mucopolysaccharidosis type IVB / 中华儿科杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHOD</b>A 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.</p><p><b>RESULT</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Comparison and clinical application of two methods for determination of plasma chitotriosidase activity / 中华儿科杂志

<p><b>OBJECTIVE</b>Chitotriosidase (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.</p><p><b>METHOD</b>Plasma 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.</p><p><b>RESULT</b>(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.</p><p><b>CONCLUSION</b>The 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.</p>

Clinical presentation and therapeutic outcomes of carnitine deficiency-induced cardiomyopathy / 中华儿科杂志

<p><b>OBJECTIVE</b>Carnitine deficiency has been associated with progressive cardiomyopathy due to compromised energy metabolism. The objective of this study was to investigate clinical features of carnitine deficiency-induced cardiomyopathy and the therapeutic efficacy of L-carnitine administration.</p><p><b>METHOD</b>Between January 2010 and December 2011, filter-paper blood spots were collected from 75 children with cardiomyopathy. Free carnitine and acylcarnitine profiles were measured for each individual by tandem mass spectrometry (MS/MS). For those in whom carnitine deficiency was demonstrated, treatment was begun with L-carnitine at a dose of 150 - 250 mg/(kg·d). Clinical evaluation, including physical examination, electrocardiography, chest x-ray, echocardiography and tandem mass spectrometry, was performed before therapy and during follow-up.</p><p><b>RESULT</b>Of 75 cardiomyopathy patients, the diagnosis of carnitine deficiency was confirmed in 6 patients, which included 1 boy and 5 girls. Their age ranged from 0.75 to 6 years. Free carnitine content was (1.55 ± 0.61) µmol/L (reference range 10 - 60 µmol/L). Left ventricular end-diastolic diameter (LVDd) was (5.04 ± 0.66) cm and left ventricular ejection fraction (LVEF) was (38.5 ± 10.5)%. After 10 - 30 d therapy of L-carnitine, free carnitine content rose to (30.59 ± 15.02) µmol/L (t = 4.79, P < 0.01). LVDd decreased to (4.42 ± 0.67) cm (t = 4.28, P < 0.01) and LVEF increased to (49.1 ± 7.6)% (t = 6.59, P < 0.01). All patients received follow-up evaluations beyond 6 months of treatment. Clinical improvement was dramatic. LVEF returned to normal completely in all the 6 patients. LVDd decreased further in all the 6 patients and returned to normal levels in 3 patients. No clinical signs or symptoms were present in any of the 6 patients. The only complications of therapy had been intermittent diarrhea in 1 patient.</p><p><b>CONCLUSION</b>Tandem mass spectrometry is helpful to diagnose carnitine deficiency and should be performed in all children with cardiomyopathy. L-carnitine has a good therapeutic effect on carnitine deficiency-induced cardiomyopathy.</p>

Primary carnitine deficiency in 17 patients: diagnosis, treatment and follow up / 中华儿科杂志

<p><b>OBJECTIVE</b>Many 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.</p><p><b>METHOD</b>We 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.</p><p><b>RESULT</b>Seventeen 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].</p><p><b>CONCLUSION</b>Seventeen 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.</p>

Application of succinylacetone levels measurement in the blood and urine in the diagnosis of tyrosinemia type 1 / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the diagnostic method of tyrosinemia type 1 and evaluate its value, the succinylacetone levels in the blood of suspected patients with tyrosinemia were tested by tandem mass spectrometry, and the succinylacetone in the urine was tested by gas chromatography-mass spectrometry.</p><p><b>METHOD</b>A total of 190 patients suspected of having tyrosinemia, were tested by tandem mass spectrometry for measurement of the level of succinylacetone in the blood, and detected by gas chromatography-mass spectrometry for measurement of the level of succinylacetone and organic acid in the urine. The method of measuring the level of succinylacetone in blood by tandem mass spectrometry as follows: After the diameter of 3 mm dry blood spots were punched into wells of 96-well plate, 100 µl 80% acetonitrile were added into each well, which contained hydrazine monohydrate and the internal standard of succinylacetone. The supernatant fluid were transferred to another 96-well plate and dried under heated nitrogen, after the plate was incubated for 30 min at 65°C. The residual hydrazine reagent was removed by addition of 100 µl methanol to each well and evaporated under heated nitrogen. The mobile phase (80% acetonitrile) was added to each well and 20 µl samples were tested by tandem mass spectrometry. The diagnostic terms were the clinical manifestation and the high level of succinylacetone in both blood and urine.</p><p><b>RESULT</b>Eleven patients were diagnosed as tyrosinemia type 1, with 9 males and 2 females. Their ages ranged from 2 months to 6 years. The succinylacetone levels in the blood of the patients were remarkably increased (7.26-31.09 µmol/L), with an average of (14.2 ± 7.8)µmol/L. Seven patients were tested for the level of succinylacetone in the urine by gas chromatography-mass spectrometry, and 4 were positive and 3 negative. Their tyrosine levels in the blood were 190-543 µmol/L(Normal: 20 - 100 µmol/L), with an average of (327.3 ± 125.8) µmol/L. All the patients presented the symptoms of hepatomegaly. Among them, 9 patients died and 2 patients were improved after treatment.</p><p><b>CONCLUSION</b>The higher levels of succinylacetone in the blood or urine is a remarkable evidence for the diagnosis of tyrosinemia type 1. Determination of succinylacetone in the dry blood spots using tandem mass spectrometry was a good method for diagnosis of tyrosinemia type 1. To test succinylacetone in urine by gas chromatography-mass spectrometry may yield a false-negative result for tyrosinemia type 1.</p>

Application of enzyme assay and gene analysis in the prenatal diagnosis for a family with glycogen storage disease type II / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To carry out prenatal diagnosis for a glycogen storage disease type II (GSD II ) affected family.</p><p><b>METHODS</b>The acid-α -glucosidase (GAA) activity was measured in whole leukocytes and cultured amniocytes with 4-methylumbelliferyl-α -D-glucopyranoside as substrate and with acarbose as inhibitor. The coding regions of GAA gene were amplified by polymerase chain reaction and analyzed by direct DNA sequencing.</p><p><b>RESULTS</b>The proband and the fetus had low GAA activity (12.3% and 1.1% of the average normal range, respectively). Mutation analysis of the GAA gene revealed a novel nonsense mutation p.W738X and a reported nonsense mutation p.E888X in both the proband and the fetus; the reported pseudodeficiency allele c.[1726G to A: 2065G to A] was found in the proband, the mother and the fetus.</p><p><b>CONCLUSION</b>The proband and the fetus were both GSD II affected. A combination of GAA activity analysis and mutation analysis is efficient for the prenatal diagnosis of GSD II. Mutation analysis should be a routine method in the prenatal diagnosis of GSD II in Asian population, where pseudodeficiency allele can cause low GAA activity in normal individuals which is relatively common in Asian.</p>