Genetic association between corneal curvature-related genes and high myopia in Chinese Han population / 中华实验眼科杂志

Background High myopia is one of the primary factors of visual impairment,and its prevention and management are researching hot topics.Corneal curvature (CC) measures the steepness of the cornea which is an important parameter leading to myopia.Genome-wide association study (GWAS) showed that several genes are associated with CC in Asian populations.However,the association of corneal curvature-related genes with high myopia is unclear up to now.Objective This study was to investigate the association between single nucleotide polymorphism (SNP) in the rs74225573 (mechanistic target of rapamycin [MTOR]),rs60078183 (cytidine/uridine monophosphate kinase 1 [CMPK1]),rs1800813 (platelet derived growth factor receptor alpha [PDGFRA]),rs11204213 (retinol binding protein 3 [RBP3]) and high myopia in Chinese Han population.Methods A prospective cohort study was performed.Four hundreds and eighty-three patients with high myopia were collected in Sichuan Provincial People's Hospital from February 2012 to August 2013,with the diopter (-10.84±4.69)D in the right eyes and (-10.35±4.67)D in the left eyes or ocular axial length of (28.15±2.27)mm in the right eyes and (27.72±2.51)mm in the left eyes.Five hundreds and nineteen normal volunteers matched in age and gender were included in the same period as controls,and all the subjects were Chinese Han people without genetic relationship.The periphery blood of 4 ml was obtained for the DNA extraction from each subject under the written informed consent.The primers of rs74225573,rs60078183,rs1800813 and rs1 1204213 were designed based on the information of NCBI website.The four SNPs were amplified by real-time PCR and genotyped by SNaPshot method.Results All the genotype frequencies of these four SNPs were in Hardy-Weinberg equilibrium (HWE).There are no significant differences in minor allele frequency (MAF) distribution of rs74225573,rs60078183 and rs11204213 between high myopia group and normal control group (rs74225573:Pag-corrected =0.935,OR =0.98;rs60078183:Page-currected =0.782,OR =1.04;rs11204213:Page-currected =0.058,OR =1.66),and the M AF of rs1800813 was significantly higher in the high myopia group than that in the normal control group (Page-currected =0.001,OR =0.64).The genotype frequency of rs74225573,rs60078183 and rs11204213 was not evidently different in additive model 1 (AB vs.BB),additive model 2 (AA vs.BB),dominant model (AA+AB vs.BB) and recessive model (AA vs.AB+BB) (all at P＞0.05),while significant differences were found in genotype frequency of rs1800813 both in additive model 1 and dominant model (additive model 1:P=0.002,OR=0.59;dominant model:P=0.001,OR=0.58).Conclusions The SNP of rs1800813 in the PDGFRA gene is associated with the pathogenesis of high myopia in the Chinese Han population,but the SNPs of rs74225573 (MTOR gene),rs60078183 (CMPK1 gene) and rs11204213 (RBP3 gene) appear to be not associated with high myopia.

Cloning of Alpha gene segments from t(6;11) translocation renal cell carcinoma and analysis of their promoter activities / 中国肿瘤临床

To construct recombinant reporter plasmids containing different Alpha gene segments and Alpha1-TFEB fusion gene and to evaluate the promoter activity of the Alpha gene. Methods:Promoter regions of the Alpha gene were predicted using a software Primer 0.5. Five Alpha gene segments with different lengths and a normal TFEB gene promoter (pTFEB) were amplified via polymerase chain reaction, and recombinant reporter plasmids containing different Alpha gene segments and a normal TFEB gene pro-moter were constructed. Liposome transfection was used to transfect these vectors into the human embryo kidney 293T cells. The pro-moter activity of the Alpha gene was evaluated via luciferase assay. Meanwhile, the recombinant Alpha1-TFEB plasmid was construct-ed and transfected into the 293T cells. The TFEB expression of the recombinant Alpha1-TFEB plasmid was then detected via Western blot. Results: Recombinant reporter plasmids containing different Alpha gene segments and pTFEB were constructed successfully. Compared with the luciferase activity of pGL3-Basic, that of the groups with Alpha1, Alpha2, Alpha3, Alpha4 and Alpha5 significantly increased (P<0.01). The luciferase activity also increased significantly in the groups with Alpha1, Alpha2 and Alpha5 compared with that of the pTFEB group (P<0.01). The TFEB expression of the pGL3-Enhancer-Alpha1-TFEB was significantly higher compared with that of the pGL3-Enhancer group. Conclusion:In t(6;11) translocation RCC, the Alpha gene has a strong promoter activity and it en-hances TFEB expression. The strongest promoter activity region is in Alpha5 with a sequence from 643 bp to 693 bp.

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/microl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63degrees C by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1alpha) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

Identification and Characterization of Gliocladium viride Isolated from Mushroom Fly Infested Oak Log Beds Used for Shiitake Cultivation

A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its mycological properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30degrees C after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25degrees C) compared to CYA and PDA (< 68 mm, 5 days at 25degrees C). Penicillate conidiophores of the fungus are hyaline, smooth walled, branching above typically in four stages, and 120~240 microm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 microm x 1.8~2.3 microm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G. viride strains.

Polymorphisms of the Reg1alpha Gene and Early Onset Type 2 Diabetes in the Korean Population / 당뇨병

BACKGROUND: The Reg gene has been reported to be expressed in regenerating islets and Reg1 protein to be up-regulated at an early stage of diabetes in mice. As human Reg1alpha is homologous with murine Reg1, we investigated whether common variants in Reg1alpha are associated with type 2 diabetes in the Korean population. METHODS: We sequenced the Reg1alpha gene to identify common polymorphisms using 24 Korean DNA samples. Of 11 polymorphisms found, five common ones (g.-385T>C [rs10165462], g.-36T>G [rs25689789], g.209G>T [rs2070707], g.1385C>G [novel], and g.2199G>A [novel]) were genotyped in 752 type 2 diabetic patients and 642 non-diabetic subjects. RESULTS: No polymorphism was associated with the risk of type 2 diabetes. However, g.-385C and g.2199A lowered the risk of early-onset type 2 diabetes, defined as a diagnosis in subjects whose age at diagnosis was 25 years or more but less than 40 years (odds ratio [OR], 0.721 [0.535 to 0.971] and 0.731 [0.546 to 0.977] for g.-385C and g.2199A, respectively) and g.1385G increased the risk of early-onset diabetes (OR, 1.398 [1.055 to 1.854]). Although adjusting for errors in multiple hypotheses-testing showed no statistically significant association between the three individual polymorphisms and early-onset diabetes, the haplotype H1, composed of g.-385C, g.1385C, and g.2199A, was associated with a reduced risk of early-onset diabetes (OR, 0.590 [0.396 to 0.877], P = 0.009). CONCLUSION: Polymorphisms in the Reg1alpha were not found to be associated with overall susceptibility to type 2 diabetes, though some showed modest associations with early-onset type 2 diabetes in the Korean population.

Mutation Screening of HNF-1alpha Gene in Korean Women with Gestational Diabetes Mellitus / 당뇨병

BACKGROUNDS: Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first detection during pregnancy and mostly caused by insulin resistance and beta-cell dysfunction like type 2 diabetes. However, autoimmune or monogenic diabetes can contribute to GDM. Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by an early age of onset and an autosomal dominant pattern of inheritance. Most MODY cases are attributable to mutations in HNF-1alpha gene, also known as MODY3. We investigated whether mutations in HNF-1alpha gene are present in Korean women with GDM. METHODS: A total of 96 Korean women with GDM who have a family history of DM were screened for mutations in the HNF-1alpha gene. We evaluated the clinical characteristics of GDM women with HNF-1alpha gene mutations. RESULTS: Five of 96 patients (5.2%) were found to have a mutation in HNF-1alpha gene. Four of those (-23C > G, 833G > A (Arg278Gln), 923C > T, IVS5 + 106A > G) were novel and one (-124G > C) in promoter region was reported in previous study. The mean age of GDM women with mutations of HNF-1alpha gene was 34 years. Four women with MODY3 gene mutations required insulin therapy during pregnancy. GDM women with MODY3 gene mutations appeared to be decreased insulin secretion (HOMA-%B) than those without mutations. CONCLUSIONS: We have found the existence of MODY3 as well as novel HNF-1alpha gene mutations in Korean women with GDM.

Association Study of Tumor Necrosis Factor-Alpha Gene Polymorphism in Schizophrenia and Bipolar Disorder / 신경정신의학

OBJECTIVES: The role of cytokines has been one of the focal points of etiologic research in major psychiatric disorders. This study was designed to analyze the polymorphism of TNF-alpha and to investigate its relationship with schizophrenia and bipolar disorder. METHODS: 241 schizophrenic patients and 89 bipolar patients diagnosed by DSM-IV criteria were included as patient groups and 125 normal poplulation from the Catholic Hemopoietic Stem Cell Information Bank (Seoul, Korea) were used as a control group. DNA was extracted from the whole blood, amplified by polymerase chain reaction, and digested by NcoI. Then the obtained RFLP of two alleles, TNFA*1 with 87 bp and 20 bp, and TNFA*2 with 107 bp were assessed. All data were analyzed by chi2 test. RESULTS: The genotype and allele distributions in patients with bipolar disorder were significantly different from those in the controls. There were no significant differences in genotype frequencies of TNFA*1/1, TNFA*1/2, and allelic frequencies of TNFA*1 and TNFA*2 between the schizophrenic patients and the controls. CONCLUSION: In the present study we observed a significant association between the TNFA*2 allele with bipolar disorder, with negative result on the association of the polymorphism of TNF-alpha gene with schizophrenia. Consecutive further studies including diverse clinical variables would be required.

Possible Association between -G308A Tumor Necrosis Factor-alpha Gene Polymorphism and Major Depressive Disorder / 신경정신의학

OBJECTIVES: The present study was to examine possible association between the -G308A tumor necrosis factor (TNF)-alpha gene polymorphism and major depressive disorder (MDD) in Korean. METHODS: 108 inpatients with MDD and 125 healthy controls participated in this study. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Genotype and allele distributions in patients with MDD (p=0.02, p=0.01, respectively), were significantly different from those of the controls. CONCLUSION: The present study suggests that the -G308A TNF-alpha gene polymorphism may have a potential role for the susceptibility to MDD in Korean population.

Gene Expression of the Somatostatin Receptors, Gi2 alpha and Pit-1 alpha in GH3 Cells Permanently Transfected with a Mutant Gs alpha Gene / 대한내분비학회지

BACKGROUND: Cyclic AMP stimulates the expression of the somatostatin (SRIF) receptor (sst1-5) and human growth hormone (GH)-secreting pituitary tumors with the gsp oncogene which increases intracellular cAMP levels, and shows a good inhibitory response of the GH to SRIF. Taken together, we hypothesized that the gsp oncogene may increase the SRIF receptor expression or and factors related to the postreceptor signal transduction of the SRIF, in order to enhance its responsiveness to SRIF. To test this hypothesis, we investigated if the gsp oncogene could increase the sst1, sst2, Gi2 alpha, and pit-1 alpha gene expression in GH3 cells. METHODS: GH3 cells were permanently transfected with the plasmid expressing Gs alpha gene, where the arginine of codon 201 was replaced with histidine. Intracellular cAMP levels and GH concentrations were measured by radioimmunoassays. Gene expressions of the sst1, sst2, Gi2 alpha, and pit-1 alpha were determined by RT-PCR. RESULTS: Intracellular cAMP levels and medium GH release were increased by 1.7 and 2.7-fold in GH3 cells expressing the gsp oncogene, respectively. In GH3 cells expressing the gsp oncogene, the sst1 mRNA levels were decreased, whereas those of the sst2, Gi2 alpha and pit-1 alpha mRNA were increased. A 4-h forskolin (10 M) stimulation remarkably increased the sst1 and sst2 mRNA levels in GH3 cells expressing wild and mutant Gs alpha . However, forskolin did not affect the Gi2 alpha and pit-1 alpha mRNA levels. In contrast, SRIF (1 M, 2 h) decreased the sst2 mRNA levels only in GH3 cells expressing the gsp oncogene. CONCLUSION: These results suggest that higher expressions of sst2, Gi2 alpha, and pit-1 alpha, induced by the gsp oncogene may be a mechanism by which gsp-positive pituitary tumors show a greater response to SRIF. The discrepancy between these and in vivo results should be explored further.

Polymorphisms of the 5'-Flanking Region of the Human Tumor Necrosis Factor-alpha Gene in Korean Patients with Crohn's Disease

PURPOSE: Recently, a key role of tumor necrosis factor (TNF) in the development of inflammatory bowel disease (IBD), especially Crohn's disease (CD), has emerged. In Japan, 3 single base pair polymorphisms in the 5'-flanking region of the TNF-alpha gene at position 1031, 863, and 857, which are related to high transcriptional promoter activity, have been identified in the Japanese CD patients. And the polymorphisms of the TNF-alpha gene at position 308, 238 have been reported in western CD patients. So, in order to find the same polymorphisms in Korean population and CD patients, the author evaluate the patients diagnosed with CD, ulcerative colitis (UC) and healthy controls (HCs). METHODS: Blood samples were obtained from 70 patients with CD, 72 patients with UC and 52 healthy controls. Polymorphisms in the TNF-alpha gene at their respective positions were analyzed by single strand conformational polymorphism (SSCP), and allele frequencies in CD and UC patients were compared with those in healthy controls. RESULTS: Allele frequencies of 1031C, 863A, and 857T in health controls were 18.3%, 8.7%, and 19.2%, respectively. Polymorphic allele frequencies of 1031C, 863A, 857T were 22.9%, 27.1%, and 24.3% in CD patients respectively. The frequencies at all 3 positions were higher in CD patients than in HCs. However, the frequency at 863A was statistically significant (P=0.000). The allele frequencies of 308A and 238A alleles were 0.7% and 3.6% in CD, 0.7% and 2.1% in UC, and 1.9% and 4.8% in HCs, respectively. The allele frequency of 1031C was significantly higher in B3 than in B2 (P=0.033). CONCLUSION: Polymorphisms of 5'-flanking region of the TNF-alpha at positions 1031 (T/C), 863 (C/A) and 857 (C/T) may be associated with susceptibility of CD.