OBJECTIVETo detect potential mutation of galactosamine-6-sulfate (GALNS) gene in a Chinese girl affected with mucopolysaccharidosis type IV A (Morquio A syndrome).

METHODSThe patient was diagnosed by assaying the activities of mucopolysaccharidosis-related enzymes in leukocytes. Potential mutation in the GALNS gene was detected with PCR and Sanger sequencing.

RESULTSThe patient was characterized by short stature, skeletal deformities, normal intelligence, and auditory dysfunction. The activities of GALNS enzymes were low. A compound heterozygous missense mutation, c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met), was detected in the GALNS gene. The mutations were respectively inherited from her father and mother. Among them, the c.1094G>T (p.Gly365Val) mutation was not reported previously.

CONCLUSIONThe mutations c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met) probably underlie the pathogenesis of the disease in our patient.

Adult ; Base Sequence ; Child ; Child, Preschool ; Chondroitinsulfatases ; genetics ; Female ; Humans ; Male ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; enzymology ; genetics ; Point Mutation

BACKGROUNDSeveral studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro.

METHODSADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay.

RESULTSSecondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05).

CONCLUSIONSSecondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

Adipocytes ; cytology ; metabolism ; Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Chondroitin ABC Lyase ; metabolism ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteoblasts ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

As the components of proteoglycans, glycosaminoglycans (GAGs) are linear polysaccharides consisting of hexose and uronic acid units linked by β-1,3-glycosidic bond. GAGs mainly distribute in extracellular matrix and on cell surfaces. They guide many biological processes, such as proliferation of cells, transmission of signals and mediation of inflammation. Because of their large molecular weights, GAGs have limited biological functions in vitro. However, the appearance of chondroitinase ABC (ChSase ABC), which can lyse polysaccharides, solves the difficulties. Based on our work, we summarized the classification and the crystal structure of ChSase ABC, as well as other recent research progress on ChSase ABCs. The separation and purification methods of ChSase ABC and construction of engineering bacteria are illustrated. The stability and immobilization are also analyzed by taking account of the characterization of ChSase ABC. Finally, problems and future prospect of the ChSase ABC study are summarized.
Bacteria ; Chondroitin ABC Lyase ; chemistry ; Extracellular Matrix ; chemistry ; Glycosaminoglycans ; chemistry ; Proteoglycans ; chemistry

OBJECTIVEMucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disease caused by a deficiency of arylsulfatase B(ARSB), which is required in the degradation of dermatan sulfate and chondroitin sulfate. The deficiency of ARSB leads to an accumulation of dermatan sulfate and chondroitin sulfate in lysosomes and gross excretion in the urine.Few articles about clinical study and ARSB gene mutation analysis of Chinese MPS VI patients were published. This study aimed to explore the clinical features and characteristics of ARSB gene in Chinese children with MPS VI.

METHODThirteen children were diagnosed as MPS VI by ARSB enzyme activity determination during the period from 2009 to 2013. Their clinical features, radiological findings and urine glycosaminoglycan (GAG) levels were retrospectively reviewed. Direct sequencing was used to identify any mutation in the ARSB gene.

RESULTThirteen children were diagnosed at the average age of (3.9 ± 2.2) years with 6 male and 7 female. All of these children presented with severe form and onset at an early age of (1.5 ± 0.8) years.Other clinical features included coarse facies, short stature, skeleton deformity, corneal clouding, hepatosplenomegaly with normal intelligence. The radiological findings in all children were characteristic of dysostosis multiplex, like abnormal development of vertebral bodies of the spine, campylorrhachia and paddle-shaped widened ribs. The MRI in case 2 showed cervical cord compression and multiple cysts degeneration in the corona radiate, cella lateralis and callosum.High urine GAG levels were detected, (307.10 ± 112.14) mg/L (Normally below 70 mg/L) and (722.28 ± 245.68) µg/mg creatinine. The ARSB enzyme activity in leukocytes was low, (13.29 ± 6.22) nmol/(mg×h) [Normal range (47-169) nmol/(mg×h)] by fluorogenic assay and (0.24 ± 0.18) U/g [Normal range (1.01-11.47) U/g] by colorimetric assay. A total of 11 mutations were identified by molecular analysis, including seven previously reported mutations (p.L72R, p.G167R, p.G303E, p.F399L, p. T442M, p.Y255X and p.R327X) and four novel mutations (p.Y175D, p.S403X, p.S464X and large deletion including ex. 2, 3). The c.1197C>G (p.F399L) mutation was the most common mutation in this study (31%).

CONCLUSIONThe severe form of MPS VI is characterized by early onset and rapid illness progression. Both the radiological findings and increased urine GAG are important clues to diagnose MPS VI.Large decrease or absence of ARSB activity is diagnostic for MPS VI.Four novel mutations of ARSB gene were identified. The reported mutation c.1197C>G (p.F399L) was the hot-spot mutation in this study.

Bone and Bones ; diagnostic imaging ; pathology ; Brain ; pathology ; Child ; Child, Preschool ; Exons ; genetics ; Female ; Glycosaminoglycans ; urine ; Humans ; Infant ; Magnetic Resonance Imaging ; Male ; Mucopolysaccharidosis VI ; diagnosis ; enzymology ; genetics ; Mutation ; N-Acetylgalactosamine-4-Sulfatase ; genetics ; metabolism ; Polymerase Chain Reaction ; Radiography ; Retrospective Studies ; Sequence Analysis, DNA

OBJECTIVEMucopolysaccharidosis (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.

METHODThirty-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.

RESULT(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.

CONCLUSIONThe 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.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; Chondroitinsulfatases ; genetics ; metabolism ; DNA Mutational Analysis ; Female ; Genotype ; Haplotypes ; Humans ; Infant ; Male ; Mucopolysaccharidosis IV ; enzymology ; genetics ; pathology ; Mutation ; Mutation, Missense ; Polymerase Chain Reaction ; Protein Conformation

OBJECTIVETo provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome.

METHODSBased on ascertained etiology of the proband and genotypes of the parents, particular mutations of the GALNS gene were screened at 10th gestational week with amplification refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing.

RESULTSDHPLC screening has identified abnormal double peaks in the PCR products of exons 1 and 10, whilst only a single peak was detected in normal controls. Amplification of ARMS specific primers derived a specific product for the fetus's gene, whilst no similar product was detected in normal controls. Sequencing of PCR products confirmed that exons 1 and 10 of the GALNS gene from the fetus contained a heterozygous paternal c.106-111 del (p.L36-L37 del) deletion and a heterozygous maternal c.1097 T>C (p.L366P) missense mutation, which resulted in a compound heterozygote status.

CONCLUSIONThe fetus was diagnosed with Morquio A syndrome and a genotype similar to the proband. Termination of the pregnancy was recommended. Combined ARMS, DHPLC and DNA sequencing are effective for rapid and accurate prenatal diagnosis for fetus with a high risk for Morquio A syndrome. Such methods are particularly suitable for early diagnosis when pathogenesis is clear. Furthermore, combined ARMS and DHPLC are suitable for rapid processing of large numbers of samples for the identification of new mutations.

Base Sequence ; Chondroitinsulfatases ; genetics ; Female ; Genetic Testing ; methods ; Humans ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; genetics ; Pedigree ; Pregnancy ; Pregnancy Complications ; genetics ; Prenatal Diagnosis ; methods ; Risk Factors

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

The aim of the study was to investigate the effect of chondroitinase ABC (ChABC) on ephrin A4 (EphA4) expression after spinal cord impairment (SCI) in rats. Adult female SD rats were randomly divided into three groups: ChABC group, normal saline (NS) group and sham group. In the ChABC and NS group, the SCI model was produced by the spinal cord hemisection. The rats in sham group received sham operation without the spinal hemisection. ChABC and NS groups were intrathecally injected with ChABC and normal saline, respectively. At different time points after SCI, injured region of spinal cord was taken out as sample. The levels of EphA4 expression were measured by immunofluorescence technique and Western blot. And the expressions of growth associated protein 43 (GAP-43) and glial fibrillary acidic protein (GFAP) were detected using double immunofluorescent staining. Immunofluorescent results showed that, compared with that in sham group, the EphA4 expression was significantly down-regulated on 1, 3 and 7 d after SCI, then up-regulated on 14 and 21 d after SCI in NS group. In ChABC group, the level of EphA4 expression was significantly less than that in the NS group during the whole time after SCI. Western blot showed an identical result to that of immunofluorescent staining. The double labeling results showed that on 3 d after SCI, the number of GFAP, glial cells marker, positive cells in NS group was lower than that in sham group, but higher than that in ChABC group. Moreover, GAP-43 was not detected in all three groups. These results suggest that ChABC can decrease the expression level of EphA4 and reduce the number of astrocytes after SCI, thus improving microenvironment of the injured region and promoting axonal growth and extension.
Animals ; Astrocytes ; pathology ; Chondroitin ABC Lyase ; pharmacology ; Ephrin-A4 ; metabolism ; Female ; Neurons ; metabolism ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; pathology ; Spinal Cord Injuries ; metabolism

OBJECTIVETo study the molecular genetic mechanism of mucopolysaccharidosis type IV A(MPS IV A), and reveal the relationship between the genotype and phenotype, and provide a basis for prenatal gene diagnosis in the future.

METHODSA preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS IV A proband. Then, mutation detection was performed on the proband and her family members with PCR and direct sequencing of the PCR products. After a novel c.1567T to G mutation was detected, Xsp I restriction enzyme digestion and amplification refractory mutation system (ARMS) fast specific identification were established to analyze the sequences of exon 14 in GALNS gene, including 110 randomly selected healthy controls, the proband and other pedigree members. At the same time, bioinformatic approaches for protein secondary, tertiary structure prediction were applied to identify the novel pathologic mutation.

RESULTSThe proband's urine GAGs test was a weak positive(± ), and a c.1567T to G heterozygous termination codon mutation in exon 14 and a c.374C to T heterozygous missense mutation in exon 4 were found. The proband was compound heterozygous of the two mutations, so was her younger sister. Her mother was a carrier with only a c.1567T to G heterozygous mutation in exon 14. Her father had a heterozygous mutation of c.374C to T in exon 4. After Xsp I restriction enzyme digestion, healthy controls had three bands including 28 bp, 120 bp and 399 bp, while the proband and her mother had four bands consisting of 28 bp, 120 bp, 148 bp and 399 bp. For amplification by ARMS specific primers, it was negative for the controls, while it was positive for the proband and the carrier. The results of protein secondary and tertiary structure prediction showed that the c.1567T to G mutation located in the stop codon, resulted in stop codon (TAG) changing to glutamic acid (GAG), with the peptide chain extending 92 amino acid residues, and secondary and tertiary protein structure change, which were not found in the controls. The result of enzyme assay showed that the activity of GALNS enzyme in the affected child was 8.3 nmol/17h/mg pr, which was obviously lower than the normal value (the normal range is 41.9-92.1 nmol/17h/mg pr).

CONCLUSIONThese results illustrate that the c.1567 T to G is a novel pathologic mutation, which is the main cause of the disease in this family.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Chondroitinsulfatases ; chemistry ; genetics ; metabolism ; Female ; Genotype ; Humans ; Infant ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; enzymology ; genetics ; Mutation ; genetics ; Pedigree ; Protein Conformation ; Sequence Alignment

Acid Etching, Dental ; adverse effects ; Chondroitin ABC Lyase ; chemistry ; Dental Bonding ; Dentin ; chemistry ; ultrastructure ; Dentin-Bonding Agents ; chemistry ; Humans ; Hydrolysis ; Matrix Metalloproteinase Inhibitors ; pharmacology ; Matrix Metalloproteinases ; metabolism ; Microscopy, Electron, Transmission ; Surface Properties