Objective
To explore the clinical manifestations, treatment and outcomes of patients with c. 482G>A(p.R161Q)variant of MMACHC gene in cblC type methylmalonic acidemia(MMA).
Methods
The clinical manifestations, mass spectrometry results, genotypes, treatment and outcomes of 75 patients with cblC type MMA carrying c. 482G>A(p.R161Q)variant were retrospectively analyzed.
Results
Of the 75 patients, 57(76%)were from newborn screening and one of them had an onset. Among the rest 18 unscreened patients, 2 were diagnosed after their full sisters′ or brothers′ diagnosis, the others were clinical patients. There were 17 clinical patients, with the medium age of onset 12 years old(10 days~26 years old). 12 late onset patients(70.6%)presented with poor academic performance, memory loss, poor expression, and decreased exercise capacity, while 5 early onset patients(29.4%)presented with convulsion and delay of development. All patients were vitamin B12-responsive. The levels of blood propionylcarnitine, the ratio of propionylcarnitine to acetylcarnitine, urinary methylmalonic acid and methyldecanoic acid, and plasma homocysteine were significantly decreased after treatment(P< 0.01). All patients diagnosed from newborn screening had normal development. However, only 3 clinical patients had a rather normal outcomes and the others remained different levels of intelligence and(or)motor dysfunction after treatment.
Conclusion
The c. 482G>A(p.R161Q)variant of MMACHC gene is associated with late onset cblC type MMA. Patients with this variant have a better response to hydroxycobalamin than other variants. The outcome of patients diagnosed from the newborn screening is good. When symptoms occur, the disability rate is often high. Therefore, newborn screening is a recommended method to prevent this disease.
Key words:
Methylmalonic acidemia; MMACHC gene; Methylmalonic acid; Propionylcarnitine
Objective
Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry (MS/MS).
Methods
A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders(LSDs)of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment.
Results
The intraassay and interassay precisions ranged from 1.7% to 11.8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999. The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L·h), 0.09 μmol/(L·h), 0.12 μmol/(L·h) and 0.16 μmol/(L·h). The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51 μmol(L·h), 1.99-22.22 μmol/(L·h), 1.68-41.59 μmol/(L·h) and 2.36-19.21 μmol/(L·h). The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPSⅠ patients can be effectively detected by this MS/MS method.
Conclusions
A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.(Chin J Lab Med, 2016, 39: 761-765)
Key words:
Lysosomal storage diseases; Tandem mass spectrometry; Dried blood spot testing; Galactosylceramidase; Alpha-Glucosidases; Alpha-Galactosidase; Iduronidase
Objectives To explore whether phenylalanine affect Cdc42,Rac1,and RhoA expression and disturb dendritic development.To determine the effects of brain-derived neurotrophic factor(BDNF) on this process.Methods Neurons were cultivated up to 3 days and then treated with 0.9 mmol / L phenylalanine or 100 ng / ml BDNF.Dendritic number were determined by morphologic analysis.Cdc42,Rac1,and RhoA protein expression were examined by Western blotting analysis.Results The number of dendrites in cultured neurons reduced two days after being treated with pheny-lalanine,while BDNF could rescue this change(P 0.01),furthermore,BDNF was found to inhibit phenylalanine-induced down-regulation of Cdc42,Rac1,and RhoA protein expression(P 0.01).Conclusions Our study indicated that the protective effect of BDNF against phenylalanine-induced neuronal injury is probably mediated by expression of Cdc42,Rac1,and RhoA.It suggested a potential neuroprotective action of BDNF in prevention and treatment of brain injury in the patients with phenylketonuria.
Objective To optimize the method of primary culture for human amniotic epithelial cells (AECs) and detect the expression of hepatocelluar specific proteins in AECs. Methods AECs were obtained by collagenase and trypsin digestion method. 10,20,40 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor were added separately to the culture media,inverted microscope was used to observe the cell growth and proliferation,and the optimal condition for primary culture of AECs was obtained. The primary cells cultured without growth factors were served as controls. The expression of hepatocelluar specific proteins such as albumin,cytokeratin-18 (CK-18),alpha-1 antitrypsin (AAT) and alpha-1 foetoprotein (AFP) in cultured cells and histological sections were detected by immunohistochemical staining. Results The AECs available were relatively pure,with only a few mesenchymal cells. EGF of 10 ng/mL was chosen as an ingredient of culture medium as the growth and proliferation of AECs significantly accelerated with 10 ng/mL EGF. The expression of albumin,CK-18,AAT and AFP was detected in amnion tissues and AECs cultured in vitro. Conclusion Collagenase and trypsin digestion method and culture medium with 10 ng/mL EGF are favourable conditions for primary culture of AECs. Hepatocelluar specific proteins are expressed in human amnion tissues and AECs cultured in vitro,indicating that AECs have some characteristics of hepatocytes.
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