Polymerase Chain Reaction Restriction Enzyme Research Articles

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Association of CLTA-4 Gene Polymorphisms with Diabetes Mellitus: A Study Based on the Han Population of Northern China.

ObjectiveThis study aimed to investigate the association of CLTA-4 gene polymorphism with Type 1 and 2 diabetes mellitus (DM) (T1DM and T2DM) in the Han population of northern China.MethodsA total of 234 Chinese Han DM patients and 187 non-diabetic subjects were included in the study. Two gene polymorphisms of the CLTA-4, including CTLA-4+49A/G (rs231775) and CTLA4-318C/T (rs5742909), were studied using polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) analysis. Chi-square analysis and Student’s t-test were performed to determine the distribution of the gene polymorphism and alleles at the locus of CTLA4-318C/T and CTLA4+49A/G among T1DM and T2DM patients with or without diabetic ketosis (DK), diabetic nephropathy (DN) or autoantibodies (Ab).ResultsOur results indicated that the distribution frequencies of genotypes and alleles at the CTLA-4+49A/G and CTLA4-318C/T loci of T1DM patients were not significantly different from those of the controls. However, the distribution frequencies of genotypes and alleles at the CTLA-4+49A/G loci of T2DM patients were significantly different from the controls (P=0.024 for genotypes and P=0.004 for A and G alleles). Besides, the A and G alleles at the CTLA4-318C/T loci of T2DM DK+ patients showed significantly different distribution frequencies compared to those of the T2DM DK patients (P=0.001).ConclusionOur data suggest that the gene polymorphisms of CTLA-4, including CTLA-4+49A/G and CTLA4-318C/T, are important predictors of DM. CTLA-4 may be a susceptibility gene for T2DM. Patients with T2DM carrying the T allele at the CTLA4-318 C/T locus are more predisposed to diabetic ketosis.

Blood Cell Parameters Combined with Inflammatory Markers in the Early Diagnosis of Pulmonary Embolism

Venous thrombosis is a semi-solid formation of blood components that coalesce in the venous system, and the pathological process of its formation is called venous thrombosis. The deep veins of the lower extremities are a common site of prevalence, and the clinical diagnosis of lower extremity deep vein thrombosis can occur independently or as a complication of other diseases. There is a clear link between inflammation and coagulation/anticoagulation, with inflammatory mechanisms upregulating pro-inflammatory factors, downregulating natural anticoagulant substances, and inhibiting fibrinolytic activity; systemic inflammation is a strong pro-thrombotic stimulus; and in vivo, natural anticoagulant substances not only prevent thrombosis, but also deter inflammatory processes. The interconnection between inflammation and coagulation plays an important role in venous thrombosis. In this study, we analyzed the relationship between inflammatory markers CRP and Fg, FVIII:C and FIX:C by measuring plasma CRP concentration, Fg level, FVIII:C and FIX:C levels in patients with DVT diagnosed by ultrasound, and explored the role and mechanism of inflammatory response and coagulation factor abnormalities and the interaction between them in the development of DVT. In this paper, human blood DNA was extracted by phenol-chloroform-isoamyl alcohol extraction, and CRP 1059G/C gene polymorphism was detected by polymerase chain reaction-restriction enzyme segment length polymorphism (PCR-RFLP) nucleotide typing technique, and the genotypes of each subject were distinguished according to the bands seen by gel electrophoresis, and the frequency of each genotype was counted. Plasma CRP concentrations were measured by immunoturbidimetric assay, FVIII:C and FIX:C levels were measured by phase I assay, and plasma Fg levels were measured by coagulation assay in 59 cases (38 males and 21 females, aged 21-82 years, mean 49.67±11.12 years) and 26 controls (17 males and 9 females, aged 32-67 years, mean 50.13±8.96 years). The above indexes were compared between the two groups, and the correlation between CRP and FVIII:C, FIX:C and Fg was analyzed. Polymerase chain reaction-restriction enzyme segment length polymorphism nucleotide typing technique was used to detect the relationship between CRP 1059G/C gene polymorphism and DVT, to further search for risk factors of venous thrombosis, thus providing new ideas for the future prevention and treatment of this disease in clinical practice.

Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in cattle, sheep, and goats from Latin America and the Caribbean: a systematic review.

This study aimed to systematically collect and appraise the scientific evidence to answer the research question: What MAP genotypes have been isolated from cattle, sheep, and goats in Latin America and the Caribbean? An electronic search was conducted on three platforms (i.e., OVID®, Web of Science®, SciELO) as well as on the proceedings of the International Colloquium on Paratuberculosis. Inclusion and exclusion criteria were defined a priori and conserved through the systematic process and only articles published in peer-reviewed journals were considered. A total of 26 articles met the definitive inclusion criteria. All were published in English, in 15 different journals, and between 1989 and 2020. The relevant articles reported the use of six different genotyping techniques (i.e., polymerase chain reaction-restriction endonuclease analysis, restriction fragment length polymorphism, type-specific-PCR, mycobacterial interspersed repetitive units-variable number of tandem repeats, multi-locus short sequence repeat, single nucleotide polymorphism) in isolates from seven countries. Genotypes found so far in the region using typing techniques were mainly C type. MIRU-VNTR mostly reported INMV 1, INMV 2, and INMV 11 subtypes, among others. MLSSR reported genotypes from four different countries, reporting nine different subtypes of which 7g–10g–4ggt was the most common for loci 1, 2, and 8, respectively. Regardless the high diversity of techniques used so far to genotype Latin American and Caribbean MAP isolates, the original question of this systematic review has been answered. In addition, a relative genetic similarity between MAP strains recovered from cattle, goats, and sheep unrelatedly of the matrix and geographic origin was identified.

Detection of DNA methylation in DBF1 gene of maize inbred W64A and mutant vp14 exposed to drought stress

DNA methylation changes correlated with environmental stresses can confer a rapid adaptation to such challenges. Dehydration-responsive element-binding factor 1 (DBF1), a member of the AP2/EREBP transcriptional factor family, is induced by drought and salinity through an ABA-dependent pathway in maize seedlings. The present investigation was carried out to study the DNA methylation status of the DBF1 gene in two different maize genotypes, an inbred line W64A and a maize mutant vp14, by the methylation-sensitive restriction enzyme–polymerase chain reaction method. Stable DNA methylation upstream of the DBF1 gene in both W64A and vp14 was observed under non-stress and drought stress conditions; simultaneously, the methylation of particular cytosines in the proximal promoter or the transcribed regions was absent.

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (MAP) in dairy cattle of Nepal

Paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an economically important, endemic in many parts of the globe, and regarded as high prevalent disease of domestic and wild animals, especially ruminants, which is manifest as chronic granulomatous enteritis with decreased milk production, with serious cases resulting in progressive emaciation and death. Understanding the genetic variability of MAP, strains are important in diagnosis, epidemiological investigation, and therefore the formation of strategies for prevention and control of the disease. Thus, this study was designed to grasp the molecular characterization of MAP isolates of Nepal, as pioneer research of this area. Total of 46 MAP isolates obtained from cattle population of three different locations of dairy pocket areas of Chitwan, Nepal were typed using IS1311 polymerase chain reaction-restriction endonuclease analysis (PCR-REA) to research the MAP genotype of Nepal. The extracted DNA samples (n=46) were analyzed for the presence of MAP specific sequences (IS900) using PCR and DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. All the DNA samples were positive for the entire three MAP specific sequences based PCRs. This study revealed that ‘Bison type’ strain is the single most prevalent MAP genotype circulating within the domestic cattle population of Nepal. IS1311 PCR-REA showed that MAP DNA samples of Nepal origin belonged to ‘Bison type’, whereas, IS1311 L2 PCR-REA method showed similarity with "Indian Bison type" and different restriction profiles of ‘Bison type’ genotype as compared to non-Indian strains. The study concludes that in Nepal, "Bison type" MAP stains was prevalent in all the MAP samples obtained from dairy cattle. These results have important epidemiological implications regarding control and prevention of paratuberculosis in Nepal.

Molecular Identification, and Characterization of Mycobacterium kansasii Strains Isolated from Four Tuberculosis Regional Reference Laboratories in Iran During 2016-2018.

BackgroundNon-tuberculous mycobacterial (NTM) infections are growing concern in many countries around the globe including Iran. Among them, Mycobacterium kansasii (M. kansasii) causes both pulmonary and extra-pulmonary infections. Despite the high prevalence of M. kansasii isolates in Iran, unfortunately little is known about the epidemiological aspects of M. kansasii infection. Hence, the aim of the present study was to investigate the molecular identification, determination of subtypes variation and geographic distribution of clinical isolates of M. kansasii isolates.MethodsIn the present study, 108 clinical pulmonary isolates suspected to NTM were collected from four Tuberculosis Regional Reference Laboratories in Iran during 2016–2018. The isolates were confirmed as NTM using conventional and molecular methods. Among them, M. kansasii isolates were subjected to rpoB gene sequencing. For determination of subtyping of M. kansasii isolates, polymerase chain reaction-restriction enzyme analysis (PCR-REA) based on the hsp65 gene was performed.ResultsBased on the rpoB gene sequence analysis, 33 (30.5%) isolates were identified as M. kansasii species, compared to 31 (28.7%) isolates using phenotypic methods. The subtype I was the most frequent subtype (n=24; 72.7%), followed by subtype II (n=8; 24.2%).ConclusionWe indicated that the rate of M. kansasii isolation with clinical significance appears to be increasing in Iran, especially in highly industrialized cities. The high rate of M. kansasii subtype I may suggest that this genotype has a particular potency for colonization, and a higher epidemiological potential for causing infection in humans. More studies are needed to provide a better understanding of the biology and pathogenicity of M. kansasii subtype I.

Genetic Analysis of EGLN1 C127S Variant in Taiwanese Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder related to nigrostriatal dopaminergic neuron degeneration and iron accumulation. As a cellular oxygen sensor, prolyl hydroxylase domain containing protein 2 (PHD2, encoded by egl-9 family hypoxia inducible factor 1, EGLN1) modifies hypoxia-inducible factor alpha (HIF-α) protein for proteasomal destruction under normoxic condition. In addition, 2-oxoglutarate- (OG-) dependent dioxygenase activity of PHD2 is involved in the oxygen and iron regulation of iron-responsive element binding protein 2 (IRP2) stability. Previously increased expression of EGLN1 was found in the substantia nigra of the parkinsonian brain. We investigated the possible role of c.380 G > C (p.C127S) of EGLN1 gene in Taiwanese patients with PD. 479 patients and 435 healthy controls were recruited. Polymerase chain reaction and BsmAI restriction enzyme analysis were applied for analysis. An association between CC genotype and reduced PD risk in the recessive model (CC vs. GG + GC) was found. Our study provides a link between EGLN1 c.380 G > C SNP and the development of PD.

Polymorphisms of T149C and T950C genes in osteoprotegerin promoter sites and the relationship between serum osteoprotegerin levels and coronary heart disease

To explore the polymorphisms of T149C and T950C gene in osteoprotectin (OPG) promoter sites and the levels of serum OPG and soluble nuclear factor-ΚB receptor activator ligand (sRANKL) and the incidence of coronary heart disease (CHD). 528 patients in Tianjin suspected of CHD and underwent coronary angiography (CAG) who admitted to the department of cardiology of Tianjin Chest Hospital from April 2017 to December 2018 were enrolled. According to the CAG results, they were divided into two groups: CHD group (n = 302) and non-CHD group (n = 226). The gender, age, history of hypertension, family history of CHD, diabetes, levels of blood lipid parameters in serum and other clinical data of patients were recorded. The levels of serum OPG and sRANKL were measured by enzyme-linked immunosorbent assay (ELISA). T149C and T950C gene polymorphisms were analyzed by polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) methods. Hardy-Weinberg genetic balance test was performed for alleles. Binomial classification multivariate non-conditional Logistic regression method was used to analyze the relationship between T149C and T950C gene polymorphisms, serum levels of OPG and sRANKL and CHD. All patients were enrolled in the final analysis. The serum level of OPG in CHD group was significantly higher than that in non-CHD group (μg/L: 1.76±0.49 vs. 1.47±0.29, P < 0.01), the serum level of sRANKL was significantly lower than that in non-CHD group (ng/L: 342.14±121.38 vs. 376.63±108.66, P < 0.05). Logistic regression analysis showed that after adjusting for age, gender, blood lipid parameters, diabetes and other factors, the increase in serum OPG level was an independent risk factor for CHD [odds ratio (OR) = 1.995, 95% confidence interval (95%CI) = 1.935-2.066, P = 0.012]. PCR-RFLP results showed that TT, TC and CC genotypes were found in T149C and T950C of OPG promoter. According to Hardy-Weinberg equilibrium test, the polymorphisms of OPG T149C and T950C accorded with Hardy-Weinberg law, achieving genetic balance with representative of the population. The frequencies of TT, TC, CC and alleles T and C in T149C genotypes of non-CHD group were 53.5%, 42.9%, 3.6%, 75.0% and 25.0%, respectively, and they were 43.1%, 50.3%, 6.6%, 68.2% and 31.8%, respectively in CHD group. There were statistically significant differences in genotype and allele frequencies between the two groups (all P < 0.05). It was shown by Logistic regression analysis that the risk of CHD in TC+CC genotype of T149C was 1.86 of TT genotype (OR = 1.86, 95%CI = 1.24-2.78, P = 0.003). It was suggested that C allele might be a susceptible gene for CHD. In non-CHD group, the frequencies of TT, TC, CC, and alleles T and C in T950C genotypes were 39.8%, 46.5%, 13.7%, 63.1% and 36.9%, respectively. They were 39.4%, 43.4%, 17.2%, 61.1% and 38.9%, respectively in CHD group. There were no significant differences in genotype and allele frequencies between the two groups (all P > 0.05). Logistic regression analysis showed that TC+CC genotype of T950C was not related with CHD. The increased level of serum OPG was closely related with CHD and could be used as a risk factor for CHD. The cases carried OPG T149C TC+CC genotype might have the risk suffering CHD. C allele is might be a susceptible gene.

Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs.

In pigs, litter size is typically defined as the total number of piglets born (TNB) or the number of piglets born alive (NBA). Increasing pig litter size is of great economic interest as a means to increase productivity. The capacity of the uterus is a critical component of litter size and may play a central role in prolificacy. In this study, we investigated litter-size-related epigenetic markers in uterine tissue from Berkshire pigs with smaller litter size groups (SLGs) and larger litter size groups (LLGs) using genome-wide bisulfite sequencing (GWBS). A total of 3269 differentially methylated regions (DMRs) were identified: 1566 were hypermethylated and 1703 hypomethylated in LLG compared to SLG. The zona pellucida binding protein (ZPBP) gene was significantly hypomethylated in the LLG promoter region, and its expression was significantly upregulated in uterine tissue. Thus, the methylation status of ZPBP gene was identified as a potential indicator of litter size. Furthermore, we verified its negative correlation with litter size traits (TNB and NBA) in whole blood samples from 172 Berkshire sows as a blood-based biomarker by a porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. The results suggest that the methylation status of the ZPBP gene can serve as a valuable epigenetic biomarker for hyperprolific sows.

Association of ICAM-1 gene variant rs5498 (1462A>G) with non-obstructive azoospermia

The blood testicular barrier (BTB) is a barrier protecting the testes from damage. It also plays an important role in spermatogenesis. The intracellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin cell adhesion molecule family and is a constituent component of the BTB. This study was carried out to investigate the association between the single nucleotide polymorphism (SNP) rs5498 in the ICAM-1 gene and non-obstructive azoospermia. A total of 100 male patients with non-obstructive azoospermia and 100 fertile males (healthy control) were included in this study. Genotyping of ICAM-1 gene rs5498 was performed using polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP). Both AG and GG genotypes were significantly more prevalent among the infertile men compared to the control group (p < 0.001). The odds ratio (OR) for the polymorphic allele (G) was 2.4 with 95% confidence interval from 1.38 to 3.19 (p < 0.001). The study shows that ICAM-1 rs5498 (1462A>G) SNP is positively associated with an increased risk for obstructive azoospermia, with the genotype AG heterozygotes showing a significantly higher frequency among infertile men.

A prognostic method for the litter size in Berkshire pigs based on DNA methylation of IGFBP4 gene

Litter size is an important trait in the pig industry. Therefore, a lot of effort has been put into improving this trait. DNA methylation is an essential epigenetic modification present in unique DNA sequences. Alterations in methylation can affect transcription and phenotypic variation. The purpose of this study was to investigate the effect of DNA methylation on litter size. Methylation-specific restriction enzymes are simple and useful tools for detecting DNA methylation status. We used a pair of methylation-sensitive isoschizomers, which have the same recognition site, HpaII and MspI. Insulin-like growth factor binding protein 4 (IGFBP4) is a key regulator of ovarian follicular development and fetal growth in eutherian mammals. In this study, we discovered that IGFBP4 was hyper-methylated in the uterus tissue of a larger litter size group using bisulfite sequencing, and validated the positive relationship between the methylation status of IGFBP4 and the total number born of pigs using the porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. We suggest that the IGFPB4 gene can be used as a prognostic biomarker for hyperprolific sows and that the PMP assay is a useful tool for methylation status screening.

Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients.

Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.

MOLECULAR SCREENING OF FOUR CANINE HEREDITARY DISEASES IN TAIWAN

Animals that have inherited a mutant gene from their parents (father and/or mother) may acquire hereditary diseases. Most mutations are autosomal recessive. Usually, hereditary diseases go unnoticed until symptoms arise. The objective of this study is to screen four canine hereditary diseases in Taiwan. The diseases include persistent Müllerian duct syndrome (PMDS) and myotonia congenita (MC) in Miniature Schnauzers, Golden Retriever muscular dystrophy (GRMD) in Golden Retrievers and glycogen storage disease type Ia (GSD Ia) in Maltese. We collected 98 samples from Miniature Schnauzers, 57 samples from Golden Retrievers and 54 samples from Maltese. All molecular tests are based on restriction fragment length polymorphism (RFLP), which consists of a genomic polymerase chain reaction (PCR) plus specific restriction enzyme digestion. Results showed that the 98 samples, which were collected from Miniature Schnauzers, were all MC wild type (WT), with 78 being PMDS WT and 20 (20%) being PMDS heterozygous type. None of the samples were MC mutant type (MT) or PMDS MT; on the other hand, no GRMD mutant gene or GSD Ia mutant gene was found in Golden Retrievers and Maltese samples.

Identification of the Infection Source of an Outbreak of Mycobacterium Chelonae Keratitis After Laser in Situ Keratomileusis

Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

The clinical effects of methylenetetrahydrofolate reductase C677T gene mutation on mild vascular cognitive impairment in patients with cerebral infarction

Objective To study the clinical effects of methylenetetrahydrofolate reductase(MTHFR)C677T gene mutation on mild vascular cognitive impairment(VCI)in patients with cerebral infarction. Methods The prospective study was conducted in 180 patients with atherosclerotic acute cerebral infarction hospitalized from June 2015 to May 2016.180 patients were divided into normal homocysteine(Hcy)group(Hcy 5.46~16.20 μmol / L, n=130)and high Hcy group(Hcy>16.20 μmol / L, n=50). MTHFR C677T genotypes were detected by polymerase chain reaction-restriction endonuclease fragment length polymorphism.The VCI state and risk factors for VCI were analyzed with Montreal Cognitive Assessment scale(MoCA)and Mini Mental State Examination(MMSE)score in patients with acute cerebral infarction on admission and 14 days, 30 days and 90 days after admission. Results MMSE and MoCA scores at 14 days, 30 days, 90 days after admission were lower in high Hcy group than in normal Hcy group(P<0.05 or P<0.01). MMSE and MoCA scores were lower in T/T genotype than in C/C genotype(P<0.05)at 14 day after admission, and lower in C/T genotype than in C/C genotype at 30 day and 90 day follow-up(P<0.05 or P<0.01). Logistic regression analysis showed that Hcy may be independent risk factors for mild VCI(OR=1.274, 95%CI: 1.027~1.264, P=0.018). Conclusions MTHFR C677T gene mutations may cause hyper-homocysteinemia, and promote the emergence of VCI in patients with cerebral infarction.MTHFR C677T gene mutations may be a genetic susceptibility factor for VCI. Key words: Brain infarction; Methylenetetrahydrofolate reductase; Cognition disorders

Impact of CYP2C19 genotypes on antiplatelet therapy among Chinese patients with acute myocardial infarction after percutaneous coronary intervention

Objective: To investigate the impact of CYP2C19 genetic variants on antiplatelet therapy in Chinese patients with acute myocardial infarction(AMI) after primary percutaneous coronary intervention(PCI), and compare the antiplatelet effect between clopidogrel and ticagrelor with various CYP2C19 genotypes. Methods: From June 2014 to May 2015, 347 patients with AMI who underwent PCI treatment at Fuwai Hospital were enrolled in this single center prospective study, 221 cases were in clopidogrel group and 126 patients were in ticagrelor group.Two candidate single nucleotide polymorphisms (SNPs) (636 and 681 loci) of CYP2C19 gene were detected by polymerase chain reaction-restriction endonuclease method.Adenosine diphosphate (ADP)-induced platelet aggregation was measured by thromboelastography (TEG) at 24-48 hours after primary PCI. Results: There were 93 non-carriers (42.1%, 93/221), and 128 carriers (57.9%, 128/221) in clopidogrel group. There were 48 non-carriers (38.1%, 48/126), 78 carriers (61.9%, 78/126) in ticagrelor group. CYP2C19 genotype frequency was similar between two groups. TEGADP was significantly lower in clopidogrel group than in ticagrelor group (63.08±27.78 vs. 78.08±25.62, P<0.001), both for non-carriers (68.16±25.17 vs. 82.79±22.45, P=0.002) and for carriers (59.37±29.06 vs. 75.15±27.15, P<0.001). TEGADP was significantly higher in non-carriers than in carriers in clopidogrel group (P=0.02) and similar between non-carriers and carriers in ticagrelor group (P>0.05). Conclusion: The CYP2C19 gene mutation is high in the patients with AMI.The effect of antiplatelet of ticagrelor is stronger than clopidogrel, and this effect is not affected by CYP2C19 gene mutations.

Cutaneous infection due to Mycobacterium immunogenum: an European case report and review of the literature

(no more than 200 words): In the last few years, the incidence of cutaneous infections caused by nontuberculous mycobacteria is increasing. Since Mycobacterium immunogenum was first described in 2001, few case reports have been described, all of them in the American continent. We report a case with cutaneous infection caused by this newly discovered NTB in Europe.A 65-year-old woman presented with a 3-months history of pruritic lesions on abdomen. Examination revealed erythematous inflammatory papules, pustules, and crusts. Three weeks later, mycobacteria were cultured from the biopsy specimen. Mycobacterium immunogenum was identified based on susceptibility test results and polymerase chain reaction (PCR) restriction enzyme analysis. Treatment with clarithromycin was started. M. immunogenum is a nontuberculous mycobacterium that was first described by Wilson et al. in 2001 as a rapidly growing variety and new species in the Mycobacterium chelonae-Mycobacterium abscessus group. PCR-restriction analysis of a 439-bp segment of the hsp65 gene and/or sequencing the species-specific region of the 16S rDNA can confirm this new species. Since the description of M. immunogenum, 8 clinical case reports have been published, most involving cutaneous infections and all of them in the American continent. We present a case of cutaneous infection caused by M. immunogenum in a Spanish woman.

Effect of MTHFR Gene Polymorphism Impact on Atherosclerosis via Genome-Wide Methylation.

BackgroundAtherosclerosis seriously threats human health. Homocysteine is an independent risk factor closely related to DNA methylation. MTHFR C667T loci polymorphism is closely associated with homocysteine level. This study aimed to investigate the relationship among MTHFR C667T loci polymorphism, genome-wide methylation, and atherosclerosis.Material/MethodsBlood sample was collected from 105 patients with coronary atherosclerosis and 105 healthy controls. Pyrosequencing methylation was used to detect LINE-1 methylation level. Polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) was used to test MTHFR.ResultsLINE-1 methylation level in the patient group was significantly lower than in the controls (t=5.007, P<0.001). MTHFR C667T genotype distribution presented marked differences in the 2 groups. TT genotype carriers had significantly increased risk of atherosclerosis (OR=3.56, P=0.009). Three different genotypes of MTHFR C667T loci showed different LINE-1 methylation level between the 2 groups (P<0.01). LINE-1 methylation level in TT and CT genotype carriers was obviously lower than in CC genotype carriers (P<0.05).ConclusionsMTHFR C667T loci polymorphism may affect atherosclerosis by regulating genome methylation level.

Development of SNP-based dCAPS markers for identifying male sterile gene tms5 in two-line hybrid rice.

Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.