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ABSTRACT: αB-Crystallin plays an important part in cataract development. A novel mutation (R11H) was previously detected by our group. In the present study, we set out to investigate the possible molecular mechanism by which the R11H mutation causes cataract. We found that the mutant αB-crystallin exhibits folding defects, decreased surface hydrophobicity and enhanced chaperone-like activity compared with the wild-type αB-crystallin. The mutant protein shows nearly the same molecular mass and thermal stability as the wild-type form. Transfection studies revealed that the R11H mutant was remarkably similar to the wild-type protein in its subcellular distribution, but has an abnormal ability to induce cell apoptosis. These results suggest that the changes in hydrophobic exposure and the abnormal ability to induce programmed cell death of the mutant protein are likely to be responsible for the onset of cataract.
Biological Chemistry 12/2010; 391(12):1391-400. · 2.96 Impact Factor
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ABSTRACT: Non-small cell cancer (NSCLC) accounts for approximately 80% of all lung cancers. Reports suggested an association between the interleukin-1beta (IL-1beta) -31 and -511 gene loci and NSCLC, but few studies took into account the effect of smoking and/or alcohol drinking on the association.
Two-hundred thirteen cases of NSCLC (aged 58.2 + or - 10.1) and 213 controls (aged 59.4 + or - 10.3y) were included in this research. Information about the smoking and drinking behaviors, dietary customs, and anamnesis were obtained from all subjects by questionnaires, and genomic DNA was extracted. IL-1beta -31 and -511 gene polymorphisms were detected using PCR-RFLP. The interactions between the genotypes and alcohol drinking/smoking were analyzed using multivariate logistic regression models.
(The T/T genotype and the T allele of the IL-1beta -31 gene were associated with higher incidence of NSCLC (P<0.05). For the IL-1beta -511 locus, no difference was found in different genotypes between the NSCLC and control groups. After the adjustment of confounding variables, such as age and gender, the binary logistic analysis showed a significant gene-environment interaction (P<0.05).
The IL-1beta -31T allele was positively associated with a risk of NSCLC, and the carriers of IL-1beta -31T/T or -511C/C would have a higher risk of suffering from NSCLC if they drank alcohol or smoke heavily.
Clinica chimica acta; international journal of clinical chemistry 10/2010; 411(19-20):1441-6. · 2.54 Impact Factor
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ABSTRACT: Certain genetic polymorphisms can lead to differences in immunity function, resulting in different clinical outcomes for hepatitis B virus (HBV) patients. The aim of this study was to investigate the association between apolipoprotein E (ApoE) gene polymorphisms and HBV infection status in northern Chinese individuals.
Genomic DNA was extracted using an improved sodium iodide (NaI) method from the peripheral blood of 270 patients with hepatitis B and 112 healthy controls. Multiplex Amplification Refractory Mutation System (Multi-ARMS) was performed to analyze ApoE gene polymorphisms with three alleles (ɛ2, ɛ3, ɛ4) in patients and controls. A chemiluminescence assay was used to detect serological markers for hepatitis B infection status.
An improved PCR system for the detection of ApoE gene polymorphisms was established successfully. The frequency of the ɛ2 allele in patients with HBV infection was higher than that of normal controls (p<0.05). The ɛ2 allele, compared with the ɛ3 and ɛ4 alleles, showed positive correlation with the different HBV infection models [odds ratio (OR)=1.735, 95% confidence interval (CI): 1.509-1.999, p<0.01; OR=1.768, 95% CI: 1.554-2.011, p<0.01]. The OR for the ApoE ɛ2 allele was 1.503 in a multivariate unconditional logistic regression model (OR=1.503, 95% CI: 1.212-1.754, p<0.01).
Our results indicated that the ApoE gene polymorphism was associated with HBV infection, and the ɛ2 allele showed positive correlation with HBV infection in northern China.
Clinical Chemistry and Laboratory Medicine 10/2010; 48(12):1803-7. · 2.15 Impact Factor
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ABSTRACT: To explore the effects of ND1 gene with 3316 G-->A mutation upon mitochondrial function and elucidate its role in the development of human diabetes.
The eukaryotic expression vector pcDNA3.1B and E. coli DH5alpha were used to construct the recombinant plasmid (pcDNA3.1B-ND1) of wild-type and 3316 G-->A mutant type ND1 gene. And the recombinant plasmids were analyzed by restriction enzyme digestion and DNA sequencing. Two siRNAs (mtND11 and mtND12) specific for human mtNDA ND1 gene were designed, synthesized and then transfected into Hela cells for silencing endogenous mtDNA ND1 gene. The gene-silencing effects were analyzed by RT-PCR, SDS-PAGE and MitoCapture mitochondrial apoptosis detection kit. Later the two types recombinant plasmids were transfected into Hela cells in which endogenous mtDNA ND1 gene was silenced. After 48 h culture, the Hela cells were collected for determination of mitochondrial proteins by SDS-PAGE.
Both mtND11 and mtND12 could decrease mtDNA ND1 expression and mtND11 caused a smaller decrease. The expression of mitochondrial protein in 3316 G-->A mutant type recombinant decreased.
The normal expression of mitochondrial ND1 gene maintain the function of mitochondrial respiratory chain and cell proliferation. The 3316 G-->A mutation in mitochondrial ND1 gene might be related to the down-regulated expression of mitochondrial protein and the diabetes mellitus pathogenesis.
Zhonghua yi xue za zhi 11/2009; 89(40):2822-6.
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ABSTRACT: The aim of this study was to reveal the genetic defect of the autosomal dominant inheritance cataract in a Chinese pedigree.
Case-control study. There were 26 individuals investigated with clinical examination in a Chinese four generations pedigree. The genome DNA of the individuals was extracted by the improved NaI method. The exons of six cataract candidate genes in 204 normal controls and 42 senile cataract patients were screened for the mutation by PCR restriction fragment length polymorphism (PCR-RFLP) methods.
The phenotype of the cataract was pulverulent nuclear cataract. A novel C/T transition at nucleotide position 827 was identified in the GJA8 gene that led to a serine to phenylalanine change in codon 276. This mutation was not found in 42 senile cataract patients and in 204 controls. Four single nucleotide polymorphisms (SNPs) were also found in a cataract candidate gene in the family members.
A novel GJA8 gene mutation was found in a Chinese autosomal dominant inheritance cataract pedigree. A substitution, C276T in GJA8 gene, was identified as the most likely causative mutation underlying the phenotype of pulverulent nuclear cataract in all affected family members.
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology 08/2009; 45(8):693-8.
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ABSTRACT: Myocilin (MYOC) gene is expressed in many ocular tissues, including the trabecular meshwork, a specialized eye tissue essential in regulating intraocular pressure. Many mutations in MYOC have been detected in primary open-angle glaucoma (POAG). We investigated whether MYOC mutations contributed to the susceptibility to POAG in a Chinese family. In a four-generation family affected with POAG, ocular examinations were performed on all members of the pedigree to determine their disease status, and 200 healthy matched controls were recruited. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to determine the mutations in MYOC. Biological software was used to analyze the corresponding proteins for missense mutations. The c.1084G>- was found, for the first time, in four of eight affected patients and in one of two patients with suspected POAG. The c.1006C>T mutation was found in two of eight patients and in one of 19 subjects who were asymptomatic. The frequencies of c.1084G>- and c.1006C>T were 12.82 and 7.69%, respectively, in patients but not in the controls. These data provide additional clues to the pathogenesis of POAG because no other mutation was detected in either group. Our results suggest that the MYOC c.1084G>- may contribute to a genetic predisposition to POAG.
Molecular Biology Reports 08/2009; 37(1):255-61. · 2.93 Impact Factor
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ABSTRACT: To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).
Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1alpha and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1alpha and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.
Dau significantly inhibited IGF-I-induced HIF-1alpha protein expression but had no effect on HIF-1alpha mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1alpha and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1alpha protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.
Dau inhibits human breast cancer angiogenesis by suppressing HIF-1alpha protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.
Acta Pharmacologica Sinica 05/2009; 30(5):605-16. · 1.95 Impact Factor
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ABSTRACT: To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework.
The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment.
Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%.
Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.
European journal of obstetrics, gynecology, and reproductive biology 04/2009; 144(1):35-9. · 1.97 Impact Factor
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ABSTRACT: Infections with hepatitis B virus (HBV) may lead to a distinct clinical outcome which is partially related to host genetic variability. Our aim was to investigate the relationships between the polymorphisms of the E-selectin gene and disease progression in a HBV-infected Chinese Han population, and also to determine the plasma soluble E-selectin (sE-selectin) levels in this population.
Genomic DNA was extracted from the peripheral blood of 367 HBV carriers and 281 healthy controls. Two polymorphisms (PstI for A561C and HphI for G98T) of the E-selectin gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Circulating sE-selectin levels were measured by specific enzyme-linked immunosorbent assay (ELISA).
The frequency of the C allele (AC or CC) of the A561C polymorphism was significantly higher in patients with liver cirrhosis (LC) compared to controls (p=0.002). There was no difference in allele distribution of the G98T polymorphism. But in patients with LC, classified according to the Child-Pugh classification, the frequency of the T carrier (GT and TT) was significantly different between Child-Pugh class A and class B plus C (p=0.009). Levels of plasma soluble E-selectin (sE-selectin) were significantly increased in HBV carriers with chronic hepatitis (CH) and LC (mean+/-SD 68.94+/-34.09 and 43.39+/-18.00 ng/mL) compared to controls (13.96+/-7.50 ng/mL) (p<0.01). In the LC subgroup, levels of sE-selectin were significantly decreased from Child-Pugh class A to class C (p<0.05). In each group, individuals with the C allele showed higher sE-selectin levels than those with the A allele (p<0.05).
This is the first report describing the association between E-selectin polymorphisms and HBV-related chronic liver diseases. Our data suggest that the A561C polymorphism of the E-selectin gene may be associated with disease progression in patients with chronic HBV infection and control the expression of plasma soluble levels, while the G98T polymorphism may be related to fibrotic severity in the Chinese population.
Clinical Chemistry and Laboratory Medicine 01/2009; 47(2):159-64. · 2.15 Impact Factor
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Chinese Journal of Chemistry 12/2008; 26(12):2207 - 2215. · 0.75 Impact Factor
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ABSTRACT: Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal dominantly inherited disease mainly caused by mutations of the gene encoding the low density lipoprotein receptor (LDLR) and Apolipoprotein (Apo) B. First the common mutation R3500Q in ApoB gene was determined using PCR/RFLP method. Then the LDLR gene was screened for mutations using Touch-down PCR, SSCP and sequencing techniques. Furthermore, the secondary structure of the LDLR protein was predicted with ANTHEPROT5.0. The R3500Q mutation was absent in these two families. A heterozygous p.W483X mutation of LDLR gene was identified in family A which caused a premature stop codon, while a homozygous mutation p.A627T was found in family B. The predicted secondary structures of the mutant LDLR were altered. We identified two known mutations (p.W483X, p.A627T) of the LDLR gene in two Chinese FH families respectively.
Molecular Biology Reports 12/2008; 36(8):2053-7. · 2.93 Impact Factor
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ABSTRACT: To investigate the effects of green tea extract (GTE) and its major component (-)-epigallocatechin-3-gallate (EGCG) on the human papillomavirus (HPV) type 16 oncoproteins (E6 and E7)-induced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human cervical carcinoma cells.
Human cervical carcinoma cells of the line C-33A were divided to 4 groups to be transiently transfected with the plasmid HA-pSG5-HPV-16E6 containing the HPV type 16 oncoprotein E6, HA-pSG5-HPV-16 E7, or empty plasmid HA-pSG5, or just exposed to the transfection reagent Lipofectamine 2000 as mock transfection control group. Western blotting and ELISA were used to detect the protein expression of HIF-1alpha and the protein expression of VEGF 18, 24, and 48 h after the transfection. Twenty-four hours after the transfection, the transfected-cells were treated with GTE (40 microg/ml) or EGCG (50 micromol/L) for 8, 16, and 24 h respectively, or treated with GTE of the concentrations of 20, 40, and 80 microg/ml or EGCG of the concentrations of 25, 50, and 100 micromol/L respectively for 16 h. The HIF-1alpha and VEGF protein levels were analyzed by Western blotting and ELISA respectively and the HIF-1alpha and VEGF mRNA levels were determined with real-time PCR.
Twenty-four hours after transfection, the HIF-1alpha protein expression of the 16E6 and 16E7 groups were significantly decreased, and the VEGF protein levels of the 16E6 and 16E7 groups were (870+/-66) and (1487+/-51) pg/ml respectively, both significantly higher than that of the empty plasmid group [(366+/-65) pg/ml, both P<0.01]. Treated by 40 microg/ml of GTE or 50 micromol/L of EGCG for 16 h, (1) the HIF-1alpha protein levels of the 16E6 and 16E7 groups were significantly decreased; (2) the VEGF protein levels of the 16E6 group were (476+/-34) and (477+/-65) pg/ml respectively, and the VEGF mRNA levels of the 16E6 group were 1.208+/-0.196 and 1.174+/-0.208 respectively, all significantly lower than those of the untreated group [(870+/-66) pg/ml and 1.805+/-0.081 respectively, all P<0.01]; and (3) the VEGF protein levels of the 16E7 group were (649+/-55) and (514+/-37) pg/ml respectively, and the VEGF mRNA levels of the 16E7 group were 1.442+/-0.136 and 1.399+/-0.064 respectively, all significantly lower than those of untreated group [(1487+/-51) pg/ml and 2.123+/-0.120 respectively, all P<0.01]. The 80 microg/ml of GTE or 100 micromol/L of EGCG showed much stronger effects on the inhibition of VEGF protein expression than 40 microg/ml of GTE or 50 micromol/L of EGCG (both P<0.01).
GTE and EGCG can remarkably inhibit the HPV-16 oncoproteins-induced expression of HIF-1alpha protein, and VEGF protein and mRNA in human cervical carcinoma cells. Moreover, GTE and EGCG decrease the VEGF protein expression dose-dependently.
Zhonghua yi xue za zhi 11/2008; 88(40):2872-7.
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ABSTRACT: To evaluate the relationship between vitamin D receptor (VDR) gene polymorphism and bone mineral density (BMD) in 213 healthy children aged 6-10 year in China.
A questionnaire survey of dietary pattern, outdoor activity was conducted among 213 children (boys 126, girls 86) randomly selected in Xishui county of Hubei province. The BMD was determined by dual energy X-ray absorptiometry at the distal forearm, calcium, phosphorus, and alkaline phosphatase in serum were immediately analyzed. The FokI polymorphism was detected by using PCR-RFLP.
BMD was significantly higher in boys than in girls in 8/9 year group. (2) the frequencies of FF, Ff, and ff genotype were 25.8%, 62.0% and 12.2%, respectively; no difference was found between boys and girls. (3) BMD of children carrying FF genotype was higher (0.256+/-0.03) than those of carrying Ff genotype (0.241+/-0.03), P<0.01; the Ff genotype was associated with lowest forearm BMD in both boys and girls. Outdoor activity also positively affected peak bone mass.
The Fok1 polymorphism of the VDR receptor seems to directly affect bone mineral mass in Chinese children.
Clinica Chimica Acta 07/2008; 395(1-2):111-4. · 2.54 Impact Factor
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ABSTRACT: In clinic, the patients with acute myocardial infarction (AMI) are at high risk to develop ischemia-induced ventricular arrhythmias leading to sudden cardiac death (SCD). Some studies suggest that individual susceptibility to ischemia-induced arrhythmia may be related to the genes encoding ion channels. One of them is the cardiac ATP-sensitive potassium channel (K(ATP)), which is an octamer composed of four pore-forming inwardly rectifying potassium-channel subunits (Kir6.2) and four regulatory sulfonylurea-receptor subunits (SUR2A). They play important roles in the physiology and pathophysiology of cardiovascular system by coupling the metabolic state of the cells to cellular electrical activity. So far, some mutations and polymorphisms of Kir6.2/KCNJ11 gene showed significant correlation with type 2 diabetes. But it was not sure whether it was associated with acute myocardial diseases. Hence a complete mutational analysis of Kir6.2/KCNJ11 gene was performed in a pedigree of sudden cardiac death. The complete coding region and the intron-exon boundaries of KCNJ11 were amplified from genomic DNA using polymerase chain reaction (PCR). Direct sequencing was done to identify any mutations and then further confirmed by restriction site polymorphism (RSP) approach. No mutation was detected in the samples analyzed, a common polymorphism K23E (A>G) was noticed in this pedigree and the proband showed a homozygote genotype (G/G). The result suggests that the Kir6.2/KCNJ11 gene is not related to sudden cardiac death in this family.
Molecular Biology Reports 06/2008; 35(2):119-23. · 2.93 Impact Factor
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ABSTRACT: To screen the mutations of the low density lipoprotein receptor (LDLR) gene in a familial hypercholesterolemia (FH) family, and analyze the LDL-uptaking function of LDLR on lymphocytes of patients.
Genomic DNA was extracted from four affected members in a Chinese FH family. The presence of apoB100 gene R3500Q mutation which results in familial defective apolipoprotein B100 (FDB) was excluded first. Fragments of the LDLR gene were amplified by touch-down polymerase chain reaction (Touch-down PCR) and analyzed by single-strand conformational polymorphism (SSCP). The suspect fragments of the LDLR gene were cloned and sequenced. Furthermore, the lymphocytes bounded with fluorescent-labeled LDL (DiI-LDL) were measured by fluorescence flow cytometry.
A nonsense mutation was identified in exon 10 of LDLR gene. This mutation gave rise to a premature stop codon (W462X), resulting in the absence of most of the LDLR domains. It was detected in all the affected members of the FH family. The ratios of functional LDLR in lymphocytes from patients and normal controls were 63.7% and 77.3% respectively. As a result, the activity of the functional LDLR in patients was just 82.4% of that in the normal controls.
It is possible that the W462X mutation of LDLR gene is the main cause for the disease in this family.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 03/2008; 25(1):55-8.
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ABSTRACT: To identify the genetic cause responsible for the autosomal dominant hereditary cataract in a Chinese family.
A whole family of a proband who has a dominant congenital pulverulent nuclear cataract was recruited into Zhongnan Hospital. The lenses of patients were observed by a slit-lamp microscope, and the lenses of the proband's mother were analyzed by scanning electron microscopy. Mutation screening was performed in the cataract candidate genes coding for crystallins and connexin 50 by sequencing of polymerase chain reaction (PCR) products amplified from blood leukocyte DNA samples of eight family members. The identified mutation was then investigated in other participated family members, 200 normal controls, and 40 senile cataract patients by the restriction fragment length polymorphism (RFLP) method.
The structure of the lens opacities of the proband's mother is puffy, and the fibers are tangled under a scanning electron microscope. A novel C>T transition at nucleotide position 827 was determined in the connexin 50 (GJA8) gene. This mutation led to a serine (S) to phenylalanine (F) amino acid substitution in amino acid position 276 where the secondary structure prediction suggested a helix replaced by a sheet. And the mutation was neither found in the 200 controls nor in the 40 senile cataract patients.
A novel GJA8 gene mutation was found to be associated with hereditary cataract in a Chinese congenital cataract family.
Molecular vision 01/2008; 14:418-24. · 2.20 Impact Factor
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ABSTRACT: To investigate the genetic linkage of primary open-angle glaucoma (POAG) in a Chinese family.
We have screened for myocilin (MYOC) gene mutations in a glaucoma family of five generations. There are fifty-six members of whom 11 were confirmed to have POAG , two with ocular hypertension were considered as POAG suspect, and the remaining 43 were asymptomatic. We also recruited 200 unrelated healthy Chinese subjects as normal control. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to identify mutations in the three exons of MYOC. Presymptomatic diagnoses were made for the family members seeking consultation based on the results of both clinical examination and genetic analysis.
Among three allelic variants identified in this pedigree (Pro13Leu [38 C-->T], Arg76Lys [227G-->A], and Gln337Stop [1009C del]), Pro13Leu and Gln337Stop were reported to be novel mutations while Arg76Lys has been previously documented. Our results show that all 11 POAG patients carry the Gln337Stop mutation and that four POAG patients and one POAG suspect (V:2) were found to have the Pro13Leu mutation. In addition, Arg76Lys polymorphism was identified in two patients and a POAG suspect (V:5).
Pro13Leu and Gln337Stop mutations of MYOC are likely responsible for the etiology of POAG in this pedigree, but the causative mechanism needs further research.
Molecular vision 01/2008; 14:1666-72. · 2.20 Impact Factor
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ABSTRACT: Mitochondria provide cells with most of the energy in the form of ATP. Mutations in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM) because ATP plays a critical role in the production and the release of insulin. To systematically determine mutant loci and to investigate their association with T2DM in Chinese Han population, 17 commonly reported mutant loci were screened in 236 cases of T2DM and 240 normal controls by PCR-RFLP, allele-specific PCR (AS-PCR) and DNA sequencing methods. Biological softwares were used to analyze the secondary structure of DNA, RNA and the corresponding proteins for missense mutations. Sixteen mutant loci were detected in total, of which five were novel, GenBank accession nos. were DQ092356, DQ473644 and DQ473645; they were mainly in16S rRNA, ND1 and ND4 gene. There was significant difference between the two groups for ND1 and ND4 genes mutation frequencies (ND1: P=0.001, OR=3.944, 95% CI 1.671-9.306; ND4: P=0.010, OR=5.818, 95% CI 1.275-26.537). No significant association was observed between the two groups for 5178A/C polymorphisms (P=0.418). Our study suggested that T3394C and A12026G might be associated with T2DM in Chinese Han population, and T2DM with mtDNA variant should be considered mitochondrial diabetes.
Diabetes Research and Clinical Practice 07/2007; 76(3):425-35. · 2.75 Impact Factor
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ABSTRACT: Plasma chitotriosidase had been proposed as a biochemical marker of macrophage accumulation in several lysosomal storage disorders. The selection of wavelength and possible interferences and errors have not yet been explored in the assay of chitotriosidase activity. We evaluated the feasibility of measurement of plasma chitotriosidase activity by fluorescence spectrophotometry and established pediatric reference values for earlier diagnosis of related diseases.
We assayed plasma chitotriosidase activity in 104 healthy Chinese children by a fluorometric approach which combines 3-dimension scan spectra, wavelength scan spectra, time scan spectra and fluorescence intensity analysis.
The optimal excitation wavelength and emission wavelength were 358 and 448 nm, respectively. A change of enzyme activity over time was observed fluorometrically, The reference value was 13.04+/-4.94 nmol/ml/h (12.45+/-4.37 nmol/ml/h for boys and 14.04+/-3.99 nmol/ml/h for girls).
We present an integrated application of the fluorescence spectrophotometry as an ideal tool to determine enzymatic activity with 4-methylumbelliferyl triacetylchitotrioside as labeled substrates in clinical laboratory. The function of 3D scan was proved powerful in determination of plasma chitotriosidase activity. The establishment of plasma chitotriosidase activity reference pediatric values was potentially useful for the evaluation of all related diseases.
Clinica Chimica Acta 05/2007; 379(1-2):134-8. · 2.54 Impact Factor
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ABSTRACT: To establish a rapid and precise high-throughput mitochondrial (mt) DNA chip and to investigate the relationship between mtDNA tRNA Leu (UUR) and ND1 gene mutations and diabetes mellitus.
A wild-type and mutant probes of 28 loci in tRNA Leu (UUR) and ND1 gene were immobilized on the Hybond N + nylon membrane by UV-crosslinking, then the mtDNA chips were used to detect 28 loci mutation in 200 cases of type 2 diabetes mellitus and 210 matched healthy controls. All the mutations were further confirmed by DNA sequencing. Mfold and Antherprot softwares were used to predict the secondary structures of the mutant gene and protein.
The mtDNA chip, which could detect 28 loci mutations, was successfully developed. In diabetic group, there were 2 (1.0%) cases of T3 290C mutation, 6 (3.0%) of G3 316A (Ala-->Thr) mutation, 5 (2.5%) of T 3 394C (Tyr-->His) mutation, 1 (0.5%) of T3 593 C (Val-->Ala) mutation, 1 (0.5%) of A3 606G (Leu-->Leu) mutation, 8 (4.0%) of A4 164G (Met-->Met) mutation, 2 (1.0%) of T4 216C (Met-->His) mutation. In the controls, 1 (0.5%) carrier of G3 316A mutation and 5 (2.4%) carriers of A4 164G mutation were found. There was significant difference between two groups for T3 394C mutation frequencies (P = 0.027). The secondary structures of the mutant proteins of G3 316A, T3 394C, T3 593C and T4 216C mutations were all different from those of the wild-types'.
mtDNA chip is a rapid and reliable high-throughput method for mutations detection, and T3 394C mutation in ND1 gene might contribute to the pathogenesis of mitochondrial diabetes.
Zhonghua yi xue za zhi 11/2006; 86(40):2853-7.
