The Etiological Study of Three Hunter Syndrome Families in Southern China / 中山大学学报(医学科学版)

ObjectiveTo reveal the molecular pathogenesis of Hunter syndrome in three families in southern China and to clarify the correlation between phenotype and genotype， so as to lay a foundation for future prenatal or preimplantation genetic diagnosis. MethodsOn the basis of initial clinical diagnosis and pedigree analysis， qualitative detection of glycosaminoglycans in urine was performed first， and then anticoagulant blood samples were collected from the children and their relatives. DNA was extracted and the IDS gene sequence was analyzed by PCR and Sanger sequencing. Various methods such as RT-PCR and bioinformatics analysis were used to identify the pathogenicity of the new variants. ResultsThe urine test results of the patients in the three families were all strongly positive（++）. Probands were all male， with hemizygous mutations in IDS gene from their mothers， and the mutation sites were c.615_622delCATACAGT， c.847_848delGT and IVS7 ds+1 G>A， respectively. The cross-species conservation analysis showed that the amino acid of IDS gene mutation site was highly conserved during species evolution. Compared with the normal protein， mutant proteins exhibited significant differences in the predicted results of advanced structure. The variants identified in the three families were classified as pathogenic by ACMG criteria. ConclusionsThe three probands were diagnosed with Hunter syndrome. The c.615_622del（p.Il206Valfs*18）， c.847_848del（p.Val283Alafs*57） and IVS7 ds+1 G>A （p.G336Dfs*12） of IDS gene are all novel pathogenic mutations， which are the underlying causes of morbidity in children. This study has further enriched the mutation spectrum of IDS gene.

Inhibitory Effect of High Concentration Insulin on K562 Cell Proliferation and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of high concentration insulin on K562 cell proliferation and its underlying mechanism.</p><p><b>METHODS</b>K562 cells were treated by different concentration of insulin and/or anti-IGF-1R antibody (IGF-1R-Ab), MTT assay and flow cytometry were used to detect the K562 cells proliferation and apoptosis, respectivety; Western blot was used to measure the expression and phosphorylation level of IGE-IR, Akt, Erk1/2 in K562 cells under the different concentration of insulin.</p><p><b>RESULTS</b>MTT assay showed that less than 40 mU/ml insulin could promote K562 cell proliferation, while high concentration (> 40 mU/ml) insulin has been shown to inhibit K562 cell proliferation; Flow cytometry showed that 40 mU/ml insulin suppressed K562 cell apoptosis (P < 0.05), while 200 mU/ml insulin could significantly induce K562 cell apoptosis (P < 0.01); 0.01 to 1.0 µg/ml IGF-1R-Ab has significantly enhanced the inhibitory and inducing effects of high concentration (> 40 mU/ml) of insulin on K562 cell proliferation and apoptosis respectively (r = 0.962, P < 0.001); Western blot showed that after K562 cells were treated with different concentrations of insulin ERK, and the p-ERK expression did not change significantly, after K562 cells were treated with 200 mU/ml insulin, the expression of IGF-1R and AKT also not were changed obviously, while the phosphorylation level of IGF-1R and AKT increased.</p><p><b>CONCLUSION</b>High concentration (>40 mU/ml) of insulin inhibits K562 cell proliferation and induces its apoptosis, and its mechanism may be related with the binding IGF-1R by insulin, competitively inhibiting the binding of IGF-1 and IGF-1R, the blocking the transduction of PI3K/AKT signal pathway.</p>

AGL gene analysis of a pedigree with glycogen storage disease type III and identification of a novel mutation / 中华儿科杂志

<p><b>OBJECTIVE</b>To reveal the molecular genetic pathogenesis of the glycogen storage disease type III (GSDIII) and to provide a prerequisite for prenatal gene diagnosis in future.</p><p><b>METHOD</b>All the coding regions as well as the border areas between exons and introns of the AGL gene and the parental relevant mutation sites were directly sequenced, so that to affirm the origin of the mutation. Then, detected novel heterozygous mutation was confirmed by cloning sequencing. Finally, definite diagnoses of the novel mutation were performed by a series of identification methods, including screening for the 100 normal controls by DHPLC in order to count the mutational frequency, analyze the conservative of the mutant amino acid sequence from 11 kinds of species and comprise the difference of the tertiary structure between the mutant protein and the normal one.</p><p><b>RESULT</b>The patient had compound heterozygous mutations, the c.100C>T (p.R34X) nonsense mutation and c. 1176_1178 del TCA deletion mutation. The p.R34X has been reported abroad, but the 1176_1178 del TCA/p.His392fs mutation is a novel one. The proband's father is heterozygous with the p.R34X mutation while his mother carries the c.1176_1178 del TCA mutation. The result from searching the dbSNP database, HGMD database and papers published in recent years showed that the c.1176_1178 del TCA is a novel mutation, but not an SNP. Conservative analysis results in 11 species indicate that the amino acid of the mutation site is highly conserved in the stage of evolution. Comparison results between the mutant protein and the normal one demonstrate that the deletion mutation results in the obvious variation of the spatial conformation of AGL protein.</p><p><b>CONCLUSION</b>The "c.1176_1178 del TCA (p.392delHis)" mutation is a novel pathogenic mutation. This mutation and the c.100C>T (p.R34X) is the cause that the proband suffer from the GSDIIIa disease. These two mutations are inherited from mother and father respectively. The methods from this paper can be used for further prenatal gene diagnosis.</p>

Rapid prenatal genetic diagnosis of a fetus with a high risk for Morquio A syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome.</p><p><b>METHODS</b>Based 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.</p><p><b>RESULTS</b>DHPLC 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.</p><p><b>CONCLUSION</b>The 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.</p>

Identification of a novel mutation of GALNS gene from a Chinese pedigree with mucopolysaccharidosis type IV A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>These 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.</p>

Effect of mastoparan-1 on lipopolysaccharide-induced acute hepatic injury in mice / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of mastoparan-1 (MP-1) on lipopolysaccharide (LPS)-induced acute hepatic injury in mice and probe into its possible mechanism.</p><p><b>METHODS</b>One hundred and four BALB/c mice were randomly divided into healthy control group (n = 8, without treatment, HC), LPS group (n = 48, with injection of LPS 5 mg/kg via tail vein), and MP-1 group (n = 48, with injection of LPS 5 mg/kg and MP-1 3 mg/kg via tail vein). Mice in LPS group and MP-1 group were sacrificed at 2nd, 6th, 12th, 24th, 48th and 72nd post injection hour (PIH), 8 mice at each time point in each group. Blood samples were collected for determination of plasma levels of LPS by kinetic turbidimetric limulus test, TNF-alpha and IL-6 by ELISA, serum levels of ALT and AST by automatic biochemistry analyzer respectively. Hepatic tissue samples were collected for determination of TLR4, TNF-alpha and IL-6 mRNA by real-time fluorescent quantitation reverse transcription polymerase chain reaction, along with the observation of pathological changes in hepatic tissue at each time point. Above-mentioned examinations were also performed in HC group.</p><p><b>RESULTS</b>Compared with those of HC group, plasma levels of LPS and TNF-alpha in LPS group significantly increased at 2nd PIH (18,320.50 +/- 2782.50 EU/mL and 988 +/- 130 ng/L, respectively), then decreased gradually to 1.80 +/- 0.80 EU/mL and 150 +/- 44 ng/L at 72nd PIH, which was close to those of HC group. The values of IL-6, ALT and AST peaked at 12th PIH, which declined to the levels close to those of HC group at 72nd PIH. Meanwhile, the expressions of TLR4, TNF-alpha and IL-6 mRNA in liver were remarkably up-regulated after injection, and the pathological changes in hepatic tissue pronounced significantly at 12th, 24th and 48th PIH. Compared with those of LPS group, the levels of LPS, cytokines, ALT and AST decreased in MP-1 group in different degrees after injection (P < 0.05 or P < 0.01), genes expression (P < 0.05 or P < 0.01) and pathological changes was respectively suppressed and alleviated in hepatic tissue.</p><p><b>CONCLUSIONS</b>MP-1 can alleviate LPS-induced acute hepatic injury in mice, which may be associated with its neutralization of LPS and attenuation of synthesis and release of inflammatory mediators.</p>

Polymyxin B antagonizing biological activity of lipopolysaccharide / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS).</p><p><b>METHODS</b>The affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR.</p><p><b>RESULTS</b>PMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages.</p><p><b>CONCLUSIONS</b>PMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.</p>

Study of lipopolysaccharide antagonizing effect of DPR-2 in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro.</p><p><b>METHODS</b>The effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR.</p><p><b>RESULTS</b>DPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells.</p><p><b>CONCLUSION</b>DPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.</p>

A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To identify the mutations of iduronate-2-sulfatase (IDS) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome.</p><p><b>METHODS</b>Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents.</p><p><b>RESULTS</b>The result showed that the patient was: DS(++), HS(++), KS(-), CS(-), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient's IDS gene. His parents' genotypes were normal.</p><p><b>CONCLUSION</b>The patient's mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder.</p>

Detection of a new mutation (1343-TT) in the iduronate-2-sulfatase gene from a Chinese patient with mucopolysaccharidosis type II / 中华儿科杂志

<p><b>OBJECTIVE</b>Mutations of the iduronate-2-sulfatase (IDS) gene is the ultimate cause of Hunter syndrome. Clarification of the nature of mutations will create a necessary premise for prenatal gene diagnosis. A mucopolysaccharidosis (MPS) type II patient and his parents from an ethnic minority in Yunnan province were studied to identify their possible mutation in IDS gene to establish the basis for prenatal gene diagnosis.</p><p><b>METHODS</b>The patient was a boy, 6 years and 10 months old. Urine glycosaminoglycans (GAGs) assay was used for preliminary diagnosis of the patient and his parents with the disease. The three related persons' DNA was extracted and the concentration and purity of the DNA were measured after the urine test results confirmed the diagnosis. Polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) analysis was performed to detect the position of the mutation around the hot spots of mutation in exon 9, 3, 8 of the IDS gene. DNA bidirectional direct sequencing was applied to analyze the mutation detected by PCR-DHPLC.</p><p><b>RESULTS</b>The results of GAGs test showed that in the child with MPS, dermatan sulfate (DS) was positive (+++), heparan sulfate (HS) (+++), chondroitin sulfate (CS) and keratan sulfate (KS) were negative (-); while in his parents none of DS, HS, CS and KS was positive. Abnormal peaks in exon 9 of IDS gene shown by PCR-DHPLC were found in the patient. His mother had heterozygotic peaks. A new frame-mutation (1343-TT) in exon 9 of IDS gene of this patient was confirmed by DNA sequencing. The position where mutation occurred was inside codon 407 (TTT), that means two "T" deleted at position 1343 base pair (1343-TT) in cDNA of the IDS gene, caused a new frame-mutation. It caused elongation of the amino acid chain to a terminal codon TGA at position 429. Thus the peptide chain was shortened from 550 to 428 amino acids. The patient is a hemizygote of the mutation and his mother is a heterozygote.</p><p><b>CONCLUSION</b>A new frame-mutation (1343-TT) on the IDS gene was identified in this study. The patient is a hemizygote and his mother is a heterozygote. The mutation (1343-TT) resulted in loss of 122 amino acids, which probably caused seriously decreased enzyme activity of IDS, and the authors speculate that this mutation may be the pathological basis of the disease. So, if the mother becomes pregnant again, a prenatal gene diagnostic test for the same mutation should be performed. Furthermore, PCR-DHPLC followed by DNA sequencing are effective methods for diagnosis, including prenatal diagnosis of MPS II.</p>

A new mutation of iduronate-2-sulfatase gene from the patient with Hunter syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutations of iduronate-2-sulfatase (IDS) gene, and to establish a basis of prenatal gene diagnosis of Hunter syndrome.</p><p><b>METHODS</b>Urine glycosaminoglycan (GAG) assay was used to preliminary diagnosis of mucopolysaccharidosis. PCR-denaturing high-performance liquidchromatograptly (PCR-DHPLC) analysis was performed to detect the mutation in exons 9, 3, 8 of the IDS gene. DNA sequencing was applied to analyze the mutation detected by PCR-DHPLC.</p><p><b>RESULTS</b>Abnormal peaks were found by PCR-DHPLC. A new frame-mutation (1569+TT) in exon 9 of IDS gene was identified by DNA sequencing. Two "T"q inserted in position 1569 base pair (1569+TT) caused a substitution of codon 482 (TTA, leucine) to 482 (TTT, phenylalanine). The "TT" insertion results in the decrease of amino acids from 550 to 482. The patient is a hemizygote and his mother is a heterozygote.</p><p><b>CONCLUSION</b>A new frame-shift mutation of IDS gene is found to report. The mutation (1569+TT) results in 68 amino acids lost. Probably it causes the enzyme activity seriously dropped down and being pathologically the basis of disease.</p>

Study of the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose (PGG) in vitro.</p><p><b>METHODS</b>The affinity of PGG with lipid A was determined with biosensor technology, and the endotoxin-neutralizing effect was assayed with LAL. Human peripheral blood mononuclear cells (hPBMC) were separated from healthy donors and cultured in vitro. The effect of different concentrations of PGG on the release of TNF-alpha and hIL-6 from LPS-stimulated hPBMC were measured by ELISA method.</p><p><b>RESULTS</b>Lipid A was combined with different concentrations of PGG. The combination speed was shortened with the increase in PGG concentration. The KD value between PGG and Lipid A was 5.2 x 10(-7) mol/L. The release of TNF-alpha and IL-6 of hPBMC under LPS stimulation (958 +/- 234 ng/L vs 1 351 +/- 99 ng/L) was obviously inhibited by PGG in the concentration of higher than 20 mg/L compared with that without PGG treatment (1 788 +/- 171 ng/L vs 1 965 +/- 232 ng/L, P < 0.05).</p><p><b>CONCLUSION</b>PGG show an anti-endotoxin effect in vitro, which may be associated with its ability to combine and neutralize endotoxin.</p>

Experimental study on the antagonistic activity of cationic multi-peptide mastoparan-1 against lipopolysaccharide / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the mechanism of cationic multi-peptide mastoparan-1 (MP-1) on the protection of mice from lipopolysaccharide (LPS) challenge.</p><p><b>METHODS</b>Thirty Kunming mice were divided randomly into MP-1, injury, protection groups with 10 mice in each group. The mice in MP-1 group were injected with 3 mg/kg MP-1 by tail vein, while those in injury group were injected with 20 mg/kg LPS by tail vein, and those in protection group 3 mg/kg MP-1 within 20 seconds after 20 mg/kg LPS injection were injected. The effects of MP-1 on the protection of mice from LPS challenge were observed. In vitro, the affinity of MP-1 and PMB to LPS was compared by biosensor and FAST fit construct and expressed as Kd. And the neutralizing activity of MP-1 and PMB in dose of 5, 10, 20, 40 micromol/L on LPS (2 microg/L) was detected by dynamic turbidimetric limulus test with LPS neutralizing 0 micromol/L MP-1 and PMB as control. The mRNA expression levels of TLR4, TNF-alpha and IL-6 in murine peritoneal macrophages (PM phi) after exposure to LPS (100 ng/ml) were assayed by RT-PCR.</p><p><b>RESULTS</b>MP-1 could significantly protect mice from LPS challenging with protection rate of 90%. In vitro, MP-1 had a high affinity (Kd value: 484.0 nmol/L) and neutralizing ability with LPS, but it was lower than that of PMB (Kd value: 18.9 nmol/L). The neutralizing effect of 20 and 40 micromol/L MP-1 was obviously stronger than that in 0 micromol/L (P < 0.01). MP could obviously inhibit the expression of TLR4, TNF-alpha and IL-6 mRNA in LPS-stimulated murine PM phi.</p><p><b>CONCLUSION</b>MP-1 can evidently protect mice from lethal LPS challenge, and the mechanism might be related to the activity of MP-1 which binding and neutralizing LPS, blocking the combination LPS with its receptors. So the murine macrophage activation induced by LPS was inhibited.</p>