Analysis of MTHFR and MTRR Gene Polymorphisms in Iranian Ventricular Septal Defect Subjects

Ventricular septal defect (VSD) is one of the most common types of congenital heart defects (CHD). There are vivid multifactorial causes for VSD in which both genetic and environmental risk factors are consequential in the development of CHD. Methionine synthase reductase (MTRR) and methylenetetrahydrofolate reductase (MTHFR) are two of the key regulatory enzymes involved in the metabolic pathway of homocysteine. Genes involved in homocysteine/folate metabolism may play an important role in CHDs. In this study; we determined the association of A66G and C524T polymorphisms of the MTRR gene and C677T polymorphism of the MTHFR gene in Iranian VSD subjects. A total of 123 children with VSDs and 125 healthy children were included in this study. Genomic DNA was extracted from the buccal cells of all the subjects. The restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) method was carried out to amplify the A66G and C524T polymorphism of MTRR and C677T polymorphism of MTHFR genes digested with Hinf1, Xho1 and Nde1 enzymes, respectively. The genotype frequencies of CC, CT and TT of MTRR gene among the studied cases were 43.1%, 40.7% and 16.3%, respectively, compared to 52.8%, 43.2% and 4.0%, respectively among the controls. For the MTRR A66G gene polymorphism, the genotypes frequencies of AA, AG and GG among the cases were 33.3%, 43.9% and 22.8%, respectively, while the frequencies were 49.6%, 42.4% and 8.0%, respectively, among control subjects. The frequencies for CC and CT genotypes of the MTHFR gene were 51.2% and 48.8%, respectively, in VSD patients compared to 56.8% and 43.2% respectively, in control subjects. Apart from MTHFR C677T polymorphism, significant differences were noticed (p < 0.05) in C524T and A66G polymorphisms of the MTRR gene between cases and control subjects.     

Association between Phosphatase Related Gene Variants and Coronary Artery Disease: Case-Control Study and Meta-Analysis

Recent studies showed that the serum alkaline phosphatase is an independent predictor of the coronary artery disease (CAD). In this work, we aimed to summarize the association between three phosphatase related single nucleotide polymorphisms (rs12526453, rs11066301 and rs3828329) and the risk of CAD in Han Chinese. Our results showed that the rs3828329 of the ACP1 gene was closely related to the risk of CAD in Han Chinese (OR = 1.45, p = 0.0006). This significant association of rs3828329 with CAD was only found in the females (Additive model: OR = 1.80, p = 0.001; dominant model: OR = 1.69, p = 0.03; recessive model: OR = 1.96, p = 0.0008). Moreover, rs3828329 was likely to exert its effect in females aged 65 years and older (OR = 2.27, p = 0.001). Further meta-analyses showed that the rs12526453 of PHACTR11 gene (OR = 1.14, p < 0.0001, random-effect method) and the rs11066301 of PTPN11 gene (OR = 1.15, p < 0.0001, fixed-effects method) were associated with CAD risk in multiple populations. Our results showed that the polymorphisms rs12526453 and rs11066301 are significantly associated with the CAD risk in multiple populations. The rs3828329 of ACP1 gene is also a risk factor of CAD in Han Chinese females aged 65 years and older.     

Risk-Association of DNMT1 Gene Polymorphisms with Coronary Artery Disease in Chinese Han Population

Recently, a significant epigenetic component in the pathogenesis of Coronary Artery Disease (CAD) has been realized. Here, we evaluated the possible association of candidate Single Nucleotide Polymorphisms (SNPs) in the epigenetic-regulatory gene, DNA methyltransferase 1 (DNMT1), with CAD in Chinese Han population. Five tag SNPs (rs16999593, rs2336691, rs2228611, rs4804494, rs7253062) were analyzed by High Resolution Melt (HRM) method in 476 CAD patients and 478 controls. Overall, there were significant differences in the genotype and allele distributions of rs2228611 and rs2336691, between patients and controls. The minor A allele of rs2228611 was associated with a lower risk of CAD (p = 0.034); modest effect in the additive analysis but also marginal significance was found in the recessive model [OR_additive = 0.404 (0.184, 0.884), p = 0.023 and OR_recessive = 0.452 (0.213, 0.963), p = 0.040] after adjusting for confounders. While the rs2336691 A allele were associated with a higher risk of developing CAD (p = 0.037); borderline significant association in both additive and dominant models [OR_additive = 1.632 (1.030, 2.583), p = 0.037 and OR_dominant = 1.599 (1.020, 2.507), p = 0.040]. In conclusion, these data provide the first evidence that occurrence of CAD may be moderated by genetic variation in the gene involved in the epigenetic machinery.     

Circulating YKL-40 Level,but not CHI3L1 Gene Variants,Is Associated with Atherosclerosis-Related Quantitative Traits and the Risk of Peripheral Artery Disease

YKL-40, a pleotropic cytokine, is emerging as a risk factor and a prognostic predictor of atherosclerotic cardiovascular disease. We attempted to elucidate the genetic, clinical and biochemical correlates of circulating YKL-40 level and, by combining it with CHI3L1 gene variants, with the risk and long-term mortality of peripheral artery disease (PAD). Plasma YKL-40 concentrations were measured in 612 Taiwanese individuals who had no clinically overt systemic disease. Clinical parameters, CHI3L1 gene promoter variants and 18 biomarker levels were analyzed. Eighty-six PAD patients were further enrolled for analysis. Significant associations were found between CHI3L1 genotypes/haplotypes and YKL-40 levels for the health examination subjects (smallest p = 8.36 × 10⁻⁷ for rs4950928 and smallest p = 1.72 × 10⁻¹⁰ for haplotype TGG) and also for PAD patients. For the health examination subjects, circulating YKL-40 level, but not CHI3L1 gene variants, were positively associated with age, smoking, and circulating levels of triglyceride, lipocalin 2 and multiple inflammatory biomarkers and negatively associated with low-density-lipoprotein cholesterol levels. Circulating YKL-40 level is also significantly associated with the risk of PAD (p = 3.3 × 10⁻²³). Circulating YKL40 level, but not CHI3L1 gene promoter variants, is associated with the risk of PAD in Taiwanese. The association of YKL-40 levels with multiple quantitative traits relating to the risk of PAD may provide a molecular basis linking YKL-40 to atherosclerotic cardiovascular disease.     

Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana

Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes.     

Identification and Characterization of Buffalo 7SK and U6 pol III Promoters and Application for Expression of Short Hairpin RNAs

RNA polymerase III (pol III) type 3 promoters, such as 7SK and U6, are routinely used to induce short hairpin RNAs (shRNAs) to knockdown gene expression by RNA interference (RNAi). To extend the application of RNAi to studies of buffalo, an shRNAs expressing system using the buffalo pol III promoters was developed. Buffalo 7SK promoter (bu7SK) and U6 promoter (buU6) sequences upstream of the full-length 7SK and U6 small nuclear RNA sequence in the buffalo genome were identified and characterized, respectively. To determine the functionality of these promoters in constructs driving shRNA expression, anti-EGFP shRNAs (shEGFP) cassettes under the direction of bu7SK and buU6 were constructed. We further compared the EGFP knockdown efficiency of constructs using bu7SK and buU6 with that of promoters of human and bovine origins in BFF cells and mouse PT67 cells by flow cytometry and quantitative real-time PCR assays. We found that the bu7SK and buU6 promoters induced the greatest level of suppression in homologous and heterologous cells relative to promoters derived from other species. Taken together, functional bu7SK and buU6 promoters were identified and characterized, thus laying the groundwork for future development of RNAi therapeutics and gene modification in buffalo species.     

Mutation in the pssA Gene Involved in Exopolysaccharide Synthesis Leads to Several Physiological and Symbiotic Defects in Rhizobium leguminosarum bv. trifolii

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3′-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.     

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.     

Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1

Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression.     

Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)_{XRCC1 GA} = 0.80, p = 0.02; OR_{OGG1 GG} = 0.70, p = 0.008; OR_{BRCA1 TT} = 0.71, p = 0.048; OR_{WRN TT} = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR_3435CT = 1.50, p < 0.0001; OR_3435TT = 1.43, p = 0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.     

Development of 30 Novel Polymorphic Expressed Sequence Tags (EST)-Derived Microsatellite Markers for the Miiuy Croaker, Miichthys miiuy

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 30 polymorphic microsatellite markers from 5053 ESTs of the Miichthys miiuy. Out of 123 EST derived microsatellites for which PCR primers were designed, 30 loci were polymorphic in 30 individuals from a single natural population with 2-13 alleles per locus. The observed and expected heterozygosities were from 0.1024 to 0.7917 and from 0.2732 to 0.8845, respectively. Nine loci deviated from the Hardy-Weinberg equilibrium, and linkage disequilibrium was significant between 22 pairs of loci. These polymorphic microsatellite loci will be useful for genetic diversity analysis and molecule-assisted breeding for M. miiuy.