Polymerase Chain Reaction And Restriction Fragment Length Polymorphism Research Articles

DNA TYPING BY TELOMERASE REVERSE GENE TRANSCRIPTASE (TERT)

The length of the telomeric DNA and polymorphism of the TERT (Telomerase Reverse Transcriptase) gene can be the basis for the development of molecular genetic markers for the productive traits of agricultural animals, in particular pigs.
 The purpose of the work was to analyze the databases of the primary structure of the pig TERT gene, to determine single nucleotide polymorphisms (SNPs), to develop a DNA system for animal typing based on the TERT gene.
 Samples of biomaterials (blood, bristles) for DNA typing were selected from the leading farms of Ukraine in groups of 4 breeds of pigs and hybrid animals. Isolation of DNA from biomaterial was carried out using the Chelex 100 reagent. Genotyping of animals by the telomerase locus was carried out on the basis of standard methods of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The alignment of nucleotide sequences during the analysis of the primary structure of the TERT gene was carried out using the MegaX software and the Blast service. Design of the structure of oligonucleotide primers for PCR was carried out using the computer program Primer3Plus.
 During the development of the method of pigs genotyping according to TERT gene, a database analysis was carried out regarding the primary structure of the gene, a region of the gene with a significant number of SNPs was identified, and oligonucleotide primers were designed for PCR amplification of this region of the gene. The study presents the conditions of amplification of the TERT gene fragment, its cleavage by restriction endonuclease RsaI at the location of SNP rs698799571, electrophoresis of the obtained restriction DNA fragments in an 8% polyacrylamide gel, and the genotypes of the animals are also determined. The developed DNA typing technique for the TERT gene was used to analyze its polymorphism in groups of 4 purebred pigs and a group of hybrid anjmals. Monomorphic homozygous TERTAA genotype was present in purebred pigs. The polymorphism of the TERT gene by SNP rs698799571 was detected in a group of pigs of the final Irish hybrid (LW x L) x Maxgro (TERTAT genotype).
 Considering the fact that studies on the telomerase gene and determination of the TERT polymorphism for SNP rs698799571 in pigs have not yet been conducted in Ukraine, the developed technique of DNA typing by the telomerase gene has a perspective for further determination of the distribution of alleles and genotypes in domestic and imported breeds, as well as in marker-associated selection.

Cholesteryl Ester Transfer Protein (CETP) I405V (rs5882) Polymorphism Affects Plasma Lipid Parameters and Lipoprotein Ratio in Hyperlipidemic Ischemic Stroke Patients

Background: High-density lipoprotein cholesterol (HDL-C) and other lipoproteins are metabolized in part by the cholesteryl ester transport protein (CETP). Cardiovascular risk and the occurrence of ischemic stroke are linked to polymorphism in the CETP gene.
 Methodology: For the study, 100 ischemic stroke patients and 100 controls with matched sexes and ages ranging from 46 to 87 were chosen. Lipoprotein ratios were computed using Excel software, and lipid parameters were evaluated using Randox diagnostic kits. Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) and 2% gel electrophoresis were used to genotype the CETP gene. The genotyping of the CETP gene were performed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) combined with 2% gel electrophoresis.
 Results: There were significant difference (P<0.0001) in the genotypic and allelic frequencies of CETP SNP between the healthy and patients with ischemic stroke. The frequencies of I/I, I/V and V/V genotypes of the CETP gene were 48%, 37% and 15% for the control and 17%, 33% and 50%, for the stroke subjects, respectively. The frequencies of I and V alleles were 67% and 33% for the control and 37.5% and 62.5% for the stroke subjects, respectively. The V allele carriers of CETP gene had higher plasma TC, TG, VLDL-C, LDL-C, Non-HDL-C, defective HDL-C, HDL2-C and HDL3-C when compared to the I allele carriers for both subjects. The V allele carriers were responsible for the increase in dyslipidemia for both subjects.
 Conclusion: The results of this study show that mutation of CETP I405V (rs5882) polymorphism causes an increased in plasma TC, TG, VLDL-C, LDL-C, Non-HDL-C, defective HDL-C, HDL2-C and HDL3-C concentration and is associated with an increased risk of ischemic stroke.

Association between vitamin D receptor gene TaqI (731236) polymorphisms and breast cancer risk in Iraqi women

Breast cancer BC is the most frequent cancer among women worldwide. Vitamin D inhibits cell proliferation through the vitamin D receptor (VDR), which may have an impact on the risk of BC. This studywas designed to association between VDR (Taq1 T/C)(rs 731236) single nucleotide polymorphism SNP with risk of BC in Iraqi women together with serum levels 25-hydroxyvitamin D [25-(OH) D] and CA 15-3. A case -control study women was carried out at Department of Biochemistry, College of Medicine, University of Baghdad, during the period from in the period from to October 2019 to January 2021. It included 95 women with period BC that had not received any chemotherapy. The phenotyping included the measurements of serum CA 15-3 and 25-(OH) D by using ELISA Kits. The genotyping analysis comprised of VDR- Taq1 (T/C) [rs731236] by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. The mean (±SD) value of serum CA15-3 and 25-(OH)D levels were significantly increase (p < 0.05), in BC women than in control. The risk of BC was significantly increased with VDR Taq 1 SNP (T/C) (rs 731236) in heterozygous and dominant genotypes in women with BC. The TC carriers (OR=1.904, 95% C.I= 1.055, 3.43, X2=4.612, p= 0.03) and dominant TC+CC (OR=1.895, 95%C.I=1.664, 3.37, X2 = 4.75, P= 0.02). Homozygous CC carriers (OR=1.798, 95%C.I = 0.382, 8.457, X2= 0.565, p= 0.45) did not observe significant difference, and border line allele frequency C allele carriers (OR=1.888, 95%C.I=0.941..3.789, X2=3.239, p= 0.07) when compared with wild TT genotype. The significant association between VDR Taq 1 SNP (T/C) (rs 731236) and plasma V D levels among BC women (p= 0.04). In Conclusion the VDR Taq 1 SNP (T/C) (rs 731236) variant could be regarded genetic risk factor for BC development. Vitamin D receptor gene-Taq1 polymorphism was associated with plasma VD.

Vitamin D Receptor Gene ApaI and TaqI Polymorphisms in Type 2 Diabetes among Saudi Population

Background: Variants of vitamin D receptor (VDR) gene have been linked to a variety of diseases, including metabolic syndrome, cancer, bone disease, and tuberculosis. The relationship between VDR gene mutations and the susceptibility of type 2 diabetes mellitus (T2DM) in different ethnic groups is yet unknown. Vitamin D and its receptor complex have a function in regulating β-cell insulin secretion as a transcription factor.
 Objectives: The goal of this study was to see if there is a link between VDR Apa1 and Taq1 polymorphisms and T2DM susceptibility in the Saudis of Makkah environ.
 Materials and Methods: DNA was separated from peripheral blood and genotyped in 110 healthy controls and 110 unrelated people with T2DM for the VDR ApaI (G/T) rs7975232 and TaqI (A/G) rs731236 single nucleotide polymorphisms (SNPs) using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique.
 Results: The distributions of genotypes and alleles of VDR ApaI and TaqI polymorphisms were statistically indifferent across the groups investigated (P >0.05).
 Conclusion: These findings showed that polymorphisms in the VDR ApaI and TaqI genes may not be linked to T2DM risk in Saudis.

Evaluating the Association of Genetic Polymorphism of Cytochrome p450 (CYP2C9*3) in Gastric Cancer Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).

Background and aimAs a distinguished system, the cytochrome P450 (CYP) enzyme superfamily is involved in the biotransformation of several endogenous and exogenous substances including drugs, toxins, and carcinogens. Reports on the role of CYP enzyme in gastric cancer (GC) from the Eastern region of India are scarce. The present study aimed to evaluate the effect of single nucleotide polymorphisms (SNP) in cytochrome P450 family 2 subfamily C member 9 (CYP2C9*3) among cases with gastric malignancy.Material and methodsThe current study is a cross-sectional observational study carried out among 113 GC cases attending the Institute of Medical Sciences and SUM Hospital, Bhubaneswar, India, and Srirama Chandra Bhanja Medical College and Hospital, Cuttack, India. Two ml of venous blood was collected from the confirmed cases of GC. The samples were subjected to genomic DNA isolation followed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP).ResultsThe prevalence of both homozygous and heterozygous mutation in GC cases is 4% and 8%, respectively. The overall association of cytochrome P450 family 2 subfamily C member 9 (CYP2C9) mutation in GC cases is 12% whereas 88% were detected as wild/standard type. The mutation CYP2C9 SNP has been seen in Helicobacter pylori-infected cases and as well as those without H. pylori infection.ConclusionsThe CYP2C9*3 genetic polymorphism might play a significant role as a risk factor for the development of gastric malignancy irrespective of H. pylori infection, among the eastern Indian population.

Association study of KCNQ1 gene rs2237892(C/T) SNP with cardiovascular diseases in Indian population

Cardiovascular disease (CVD) is ranked highest among non-communicable diseases and considered as main cause of deaths all over the world. Genetic variants of various genes have been reported strongly associated with risk of CVDs. KCNQ1 gene which has been found to be linked with diabetes mellitus, also plays an important role in cardiac functioning. Genetic variation in KCNQ1 gene can affect the normal functioning of heart. Therefore, this study was conducted to evaluate the association between KCNQ1 gene variant rs2237892: C > T and CVD. The genotyping of genetic variant rs2237892: C > T of KCNQ1 gene was done in 100 angiography-proven CVD patients and 91 healthy controls by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) method. The distribution of genotypes was found significantly different in both case and control subjects (p < 0.05). The CC genotype of rs2237892 SNP was found to be correlated with the increased level of Triglycerides (TG) in CVD cases (p- value- 0.007). Additionally, genetic model (CC vs. CT + TT) reveals a link between the C allele and the risk of cardiovascular disease (OR – 3.671, CI: 2.012–6.696, p-value – <0.001). The present study concludes rs2237892: C > T genetic variant, C allele as the pre-disposing factor for development of CVD in the studied population of India.

Influence of the codon 72 polymorphism of the TP53 gene in the susceptibility of female cancers in Widou Thiengoly

Genetic factors have been implicated in the predisposition to cancer in Senegalese women. The arginine allele at codon 72 of exon 4 of the TP53 gene has been associated with a predisposition to several cancers in different populations. The objective of this study was to determine the effect of this polymorphism in a Senegalese population located in the locality of Widou Thiengoly. Two hundred and six (206) women living in Widou were recruited as well as thirty-two (32) control women, all assumed to be healthy. Genotyping of codon 72 of exon 4 of the TP53 gene was performed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) in 122 women. A significant association was found in the distribution of allelic and genotypic frequencies depending on the locality (p value: 5.6 x 10- 09; OR: 0.015; IC95: 3.5e-04-11e-02). However, no correlation was found between the risk factors (age, menopause, oral contraception, parity, gestational age, date of onset of first menstrual period, marital status) and the distribution of the allele frequency in the Widou locality. Due to the strong presence of the Arg allele in the Widou locality, a vast screening campaign for female cancers would be recommended.

Multiple insecticide resistance mechanisms in urban population of Anopheles coluzzii (Diptera: culicidae) from Lagos, South-West Nigeria

Malaria is a major public health challenge in Africa with Nigeria accounting for the highest burden of the disease in the world. Vector control has proved to be a highly effective component of malaria control, however, the development and spread of insecticide resistance in major vectors of malaria have been a major challenge. This study assessed resistance mechanisms in Anopheles coluzzii populations from Kosofe, Lagos mainland and Ojo Local Government Areas in Lagos, Nigeria where An. gambiae s.l is resistant to DDT and Permethrin. WHO susceptibility bioassay test was used in determining resistance status of An. coluzzii to discriminating doses of DDT and Permethrin while synergist assay was used to assess the involvement of monooxygenases in resistance development. Sub-species of An. gambiae s.l (An. gambiae and An. coluzzii) were identified using polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (PCR-RFLP) while Allele-Specific Polymerase Chain Reaction (AS-PCR) assay was used to detect knockdown mutation (kdr-West; L1014F). Biochemical assays were used in determining the activities of metabolic enzymes. High DDT resistance was recorded in An. coluzzii populations from the three sites. Mortality rate of mosquitoes exposed confirmed Permethrin resistance in Kosofe (50%) and Lagos mainland (48%) but resistance was suspected in Ojo (96%). All specimens tested were confirmed as An. coluzzii with low kdr frequency; 11.6%, 16.4% and 6.7% in Kosofe, Lagos mainland and Ojo respectively. Pre-exposure to synergist (PBO) before exposure to Permethrin led to increased mortality in all populations. Esterase activity was insignificantly overexpressed in Kosofe (p = 0.849) and Lagos mainland (p = 0.229) populations. In contrast, GST activity was significantly lower in populations from Lagos mainland (63.650 ± 9.861; p = 0.007) and Ojo (91.765 ± 4.959; p = 0.042) than Kisumu susceptible strains (120.250 ± 13.972). Monooxygenase activity was higher in Lagos mainland (2.371 ± 0.261) and Ojo (1.361 ± 0.067) populations, albeit significantly in Lagos mainland (p = 0.007) only. Presence of target-site mutation in all populations, increased mortality with pre-exposure to PBO and elevated monooxygenase in Lagos mainland population were confirmed. Multiple resistance mechanisms in some urban populations of An. coluzzii from Lagos, Nigeria calls for appropriate resistance management strategies.

Polymorphism of TLR1, TLR4, and SLC11A1 genes in populations of different cattle breeds of Ukrainian selection

Aim. To study the specificities of population genetic structure of Ukrainian Black-and-White dairy breed, Ukrainian Red-and-White dairy breed, and Ukrainian Grey cattle breed by polymorphism of TLR1, TLR4, and SLC11A1 genes. Methods. The study was conducted using the method of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results. The results of the study demonstrated that in all the experimental populations, the locus TLR4 by mutations 8732G&gt;A, 8834G&gt;C, and 2021C&gt;T was monomorphic, while loci TLR1 (1596G&gt;A) and SLC11A1 (7400C&gt;G and 7808A&gt;T) were polymorphic. For the TLR4 gene by 8732G&gt;A mutation, only individu- als with genotype BB were found; by 8834G&gt;C – with genotype GG; by 2021C&gt;T – with genotype CC. By BclI- polymorphism in the first exon of TLR1 (1596G&gt;A) the A and G allelic variants were found in each experimental population. The deviation from the Hardy-Weinberg equilibrium state was revealed for the populations of Ukrainian Black-and-White and Ukrainian Grey cattle breeds. A considerable excess of heterozygous individuals was fixed in both cattle populations (31 and 39 % respectively). Considering PstI-polymorphism in exon 11 of SLC11A1 gene, allelic variants C and G were found for SNP5 (7400C&gt;G), as well as A and T for SNP6 (7808A&gt;T) in all experimen- tal populations. As for SNP5 (7400C&gt;G), a considerable excess (from 15 to 30 %) of heterozygous individuals was determined for all experimental groups. Unlike other breeds, there was no excess (Fis = 0,04) of heterozygotes for SNP6 (7808A&gt;T) in the population of Ukrainian Grey cattle. Conclusions. The parameters of genetic variability in the different cattle populations of Ukrainian selection by TLR1, TLR4, and SLC11A1 loci were studied. The results of the study showed the impossibility of using TLR4 locus by 8732G&gt;A, 8834G&gt;C and 2021C&gt;T mutations in the programs of marker-associated selection of the investigated cattle breeds due to its monomorphic nature. The analysis of the allele and genotype distribution by TLR1 and SLC11A1 loci (presence of individuals with different genotypes in all experimental cattle breeds) indicates the possibility of using different allelic variants of these genes in the breeding programs for the studied cattle populations.

A Case-control Study on the Association of Mitochondrial Transcription Factor a Gene +35G/C Polymorphism and Mitochondrial DNA Copy Number with the Risk of Endometriosis in Indian Women

Aim: The Mitochondrial transcription factor A (TFAM) and mitochondrial (mt) DNA copy number variations are known to contribute in disease development. Genetic factors play an important role in the development of endometriosis. Therefore, this case–control study aimed to analyze the association of TFAM+35G/C polymorphism and mitochondrial copy number with the risk of endometriosis in Indian women.&#x0D; Study Design: This study was carried out on 418 subjects including 200 endometriosis cases and 218 controls.&#x0D; Methodology: Genotyping of TFAM +35G/C polymorphism (rs1937) was carried out by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Quantification of mtDNA copy number was carried out using a real time quantitative polymerase chain reaction (qRT-PCR).&#x0D; Place and Duration of Study: Department of Biochemistry, Osmania University, 2014 to 2020.&#x0D; Results: TFAM genotype as well as allele distributions were all in Hardy-Weinberg equilibrium. The results indicated a significant reduction of GG genotype frequency (P=0.009), high ‘C’ allele frequency (P=0.017) and significantly decreased mtDNA copy number in endometriosis cases compared to controls (P= 0.0001).&#x0D; Conclusion: Present study revealed a statistically significant association of decreased GG genotype of TFAM +35G/C polymorphism and mtDNA copy number with the risk of developing endometriosis in Indian women.

Paraoxonase 1 (PON1) L55M and Q192R polymorphisms are not associated with chronic kidney disease in Thai individuals with type 2 diabetes.

Decreased paraoxonase 1 (PON1) activity and PON1 polymorphisms have been found to be associated with chronic kidney disease (CKD) in type 2 diabetes mellitus (T2DM). This study aimed to investigate the association of the PON1 L55M and Q192R polymorphisms with CKD in T2DM, as well as their relationship with PON1 activity. A total of 166 T2DM patients, including 83 CKD patients and 83 non-CKD patients, were recruited. Biochemical parameters and paraoxonase (PONase) and arylesterase (AREase) activities were measured. The PON1 L55M and Q192R polymorphisms were analysed by a polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Data were analysed using the chi-square test, Student's t-test and logistic regression analysis. Total cholesterol, TGs, LDL-C and Cr were significantly higher in CKD patients than in non-CKD patients. In contrast, the estimated glomerular filtration rate (eGFR) and AREase activity were significantly lower in CKD patients than in non-CKD patients (P<.05). The genotype and allele frequencies of the PON1 L55M and Q192R polymorphisms were not significantly different between CKD and non-CKD patients. Multivariate logistic regression analysis showed no association between the PON1 L55M and Q192R polymorphisms and CKD in T2DM. In addition, among all patients, patients with the PON1 LM genotype had significantly lower PONase activity than those with the LL genotype (P<.05). Among all patients, CKD patients and non-CKD patients, those with the PON1 RR genotype had significantly higher PONase activity but lower AREase activity than patients with the QR and QQ genotypes (P<.05). PON1 activity was influenced by the PON1 L55M and Q192R polymorphisms. However, the PON1 L55M and Q192R polymorphisms may not be considered genetic biomarkers for CKD in T2DM.

Association of the Uncoupling Protein 2-866 G/A Polymorphism with Family History and Duration of Tobacco Use Disorder in a Turkish Population.

A variety of substances cause neurotoxicity by increasing intracellular oxidative stress, followed by mitochondrial dysfunction. Uncoupling proteins (UCPs) act as membrane transport proteins and reduce reactive oxygen products and mitochondrial calcium influx. We aimed to study UCP2-866 G/A gene polymorphism in tobacco use disorder (TUD) by comparing genotype distributions between TUD patients and healthy controls considering clinical parameters. One hundred eighteen patients with TUD and 96 healthy volunteers were included in the study. The diagnosis of the patients were then confirmed, based on the DSM-5 criteria. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used to determine UCP2 gene polymorphism. Our results demonstrated that the UCP2 genotype distribution and allele frequencies of the TUD patient group were significantly different from those of the control group. When the UCP2 genotype and the allele frequency distributions were compared between the two groups according to the family history of TUD in the patient group, the UCP2 genotype and allele frequency distributions were significantly different. The GG genotype or G allele percentage was significantly higher in patients with a family history of TUD, than the patients without a family history of TUD. Comparing clinical parameters based on the UCP2 genotype, the disorder's duration was significantly different between the groups of UCP2 genotype. The duration of TUD was significantly shorter in patients with GG genotype than other genotypes. In summary, the UCP2-866 G/A gene polymorphism might be associated with family history and duration of TUD in Turkish patients.

Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8.

Leishmania are intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-β and TNF signalling pathways. Here, we investigated whether MIRNA146A rs2910164 and MIRNA499 rs3746444 variants are associated with the development of L. guyanensis (Lg)-cutaneous leishmaniasis (CL). The variants MIR146A rs2910164 and MIR499A rs3746444 were assessed in 850 patients with Lg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the BioPlex assay. Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9-1.8]; Padjage/sex 0.14) compared to individuals with the genotype GG (ORadjage/sex = 0.77 [95%CI 0.56-1.0]; Padjage/sex 0.14) if exposed to Lg-infection. Heterozygous GC individuals also showed lower odds of developing CL (ORadjage/sex = 0.77 [95%CI 0.5-1.1]; Padjage/sex 0.09). Homozygosity for the allele C is suggestive of an association with the development of Lg-CL among exposed individuals to Lg-infection. However, the odds of developing CL associated with the CC genotype was evident only in male individuals (ORadjage = 1.3 [95% CI = 0.9-2.0]; Padjage = 0.06). Individuals homozygous for the G allele tend to have higher plasma IL-8 and CCL5. Similarly, for the MIR499A rs3746444, an association with the G allele was only observed among male individuals (OR = 1.4 [1.0-1.9]; P = 0.009). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 40% elevated odds of developing Lg-CL (ORadjage = 1.4 [1.1-1.9]). Individuals with the GG genotype have higher odds of developing Lg-CL (ORadjage/sex = 2.0 [95%CI 0.83-5.0]; Padjage = 0.01. Individuals homozygous for the G allele have higher plasma IL-8. Genetic combinations of both variants revealed that male individuals exposed to Lg bearing three or four susceptible alleles have higher odds of developing Lg-CL (OR = 2.3 [95% CI 1.0-4.7]; p = 0.017). Both MIR146A rs2910164 and MIR499A rs3746444 are associated with the development of Lg-CL and this association is prevalent in male individuals.

Association of eNOS (G894T, rs1799983) and KCNJ11 (E23K, rs5219) gene polymorphism with coronary artery disease in North Indian population.

Endothelial nitric oxide synthase (eNOS) and potassium voltage-gated channel subfamily J member 11 (KCNJ11) could be the candidate genes for coronary artery disease (CAD). This study investigated the relationship of the eNOS (rs1799983) and KCNJ11 (rs5219) polymorphisms with the presence and severity of CAD in the North Indian population. This study included 300 subjects, 150 CAD cases and 150 healthy controls. Single nucleotide polymorphism was evaluated by Polymerase chain reaction and Restriction fragment length polymorphism (PCR-RFLP). Analysis was performed by SPSS (version 21.0). We observed that KK genotype of KCNJ11E23K (rs5219) polymorphism (P=0.0001) genotypes and K allele (P=0.0001) was found to be a positive risk factor and strongly associated with CAD. In the case of eNOSG894T (rs1799983) there was no association found with CAD. These results illustrate the probability of associations between SNPs and CAD although specific genetic polymorphisms affecting ion channel function and expression have still to be clarified by further investigations involving larger cohorts.

Effect of Single Nucleotide Polymorphism rs1044925 in Acyl-CoA: Cholesterol Acyltransferase-1 Gene on Plasma Lipid Parameters in Patients with Ischemic Stroke

Acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) catalyzes the synthesis of cholesteryl esters from cholesterol and fatty acyl-CoA in tissues and the enzyme plays a major role in atherosclerosis and cellular cholesterol homeostasis. The study shows the effect of single nucleotide polymorphism rs1044925 in acyl-CoA:cholesterol acyltransferase-1 gene on plasma lipid parameters in patients with ischemic stroke. 100 patients with ischemic stroke and 100 controls matched for sex and aged 46-87 were selected for the study. Lipid profiles were measured using Randox kits and lipoprotein ratios were calculated using Excel software. The genotyping of the acyl-coenzyme A: cholesterol acyltransferase-1 rs1044925 SNP were performed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) combined with 2% gel electrophoresis. There were significant difference (P&lt;0.05) in the genotypic and allelic frequencies of ACAT-1 rs1044925 SNP between the normolipidemic and patients with ischemic stroke. The frequencies of AC, CC and AA genotypes of the ACAT-1 gene were 33%, 3% and 64% for the control and 57%, 5% and 38%, for the stroke subjects respectively. The frequencies of C and A alleles were 19.50% and 80.50% for the control and 33.50% and 66.50% for the ischemic stroke subjects (P &lt; 0.0001) respectively. The effects of genotypes on plasma lipid profiles and lipoprotein ratios were found for both control and stroke subjects. The A allele carriers of ACAT-1 rs1044925 SNP had lower plasma TG, TC, VLDL-C and other lipid parameters as compared to the C allele carriers for both subjects. The C allele carriers were responsible for the increase in dyslipidemia for both subjects. The results of this study show that the polymorphism of rs1044925 in the ACAT-1 gene as effect on plasma lipid profiles and lipoprotein ratios in ischemic stroke patients.

Polymorphisms in xenobiotic metabolism-related genes in patients with hepatocellular carcinoma: a case–control study

This study was performed to investigate the relationship between polymorphisms in microsomal epoxide hydrolase (mEH; Tyr113His and His139Arg substitution) and glutathione S-transferase (GST; GSTM1 deletion, GSTT1 deletion, and GSTP1.Ala114Val substitution) and their correlation with clinico-histopathological features in hepatocellular carcinoma (HCC). We evaluated environmental risk factors and genetic alterations in 556 individuals (86 cases and 470 controls). PCR multiplex for GSTM1 and GSTT1, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for GSTP1, and real-time PCR for mEH were performed. Statistical analyses were performed using multiple logistic regression tests. Age over 48 years (p < 0.001) and alcohol consumption (p = 0.021) were the predictors of increased risk of developing HCC. GSTP1.Ala114Val for all regression models (p < 0.05), except the recessive model, and the GSTT1 null genotype (odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.21–0.87, p = 0.019) were predictors of an increased risk of developing HCC. Polymorphic GSTT1, GSTM1, GSTP1.Ala114Val, and mEH.His139Arg and wild-type mEH.Tyr113His (OR = 5.04; 95% CI = 1.59–16.04; p = 0.006) were associated with HCC. Age over 48 years, alcohol consumption, and the presence of polymorphic variants of GSTP1 and GSTT1 were associated with the risk of developing HCC.

Plasma cholinesterase activity is influenced by interactive effect between omethoate exposure and CYP2E1 polymorphisms

The aim of this study was to explore the association between metabolizing enzyme gene polymorphisms and the decrease in cholinesterase activity induced by omethoate exposure. A total of 180 workers exposed to omethoate over an extended period were recruited along with 115 healthy controls. Cholinesterase activity in whole blood, erythrocyte, and plasma was detected using acetylthiocholine and the dithio-bis-(nitrobenzoic acid) method. Six polymorphic loci of GSTT1(+/-), GSTM1(+/-), GSTP1 rs1695, CYP2E1 rs6413432, CYP2E1 rs3813867, and PON2 rs12026 were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The gene-environment interactions were analyzed using the generalized linear model method. The cholinesterase activity of erythrocyte and plasma in the exposure group was significantly lower than that in the control group (P < 0.001) in general. The plasma cholinesterase activity in the TT + AT genotype in CYP2E1 rs6413432 was lower than that in the AA genotype in the exposure group (P = 0.016). Interaction between the AA genotype in CYP2E1 rs6413432 and omethoate exposure had a significant effect on plasma cholinesterase activity (P = 0.079). The decrease in plasma cholinesterase activity was associated with interaction between the AA genotypes in rs6413432 and omethoate exposure.

High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique.

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

Cryptic species diversity and molecular diagnosis of Channa orientalis; an endemic freshwater fish of Sri Lanka

Fish genetic resources and diversity are very important aspects of environmental management and fisheries and are vital for making decisions on their commercial exploitation as well as conservation. The snakehead fishes in the world have significant economic importance as food and ornamental fish. A clear understanding of species’ taxonomic status and genetic diversity is important for the utilization and implementation of conservation and management practices. Channa orientalis is a snakehead endemic to Sri Lanka that is heavily utilized in the ornamental fish export trade. Its genetic diversity has not yet been fully understood and it is difficult to distinguish it from closely resembling species. Therefore, we examined the genetic diversity of C. orientalis and developed a DNA-based marker that permits accurate, low cost, and reliable identification of C. orientalis. Determination of genetic diversity was mainly carried out through genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene. The development of the DNA-based marker for the identification of C. orientalis was done through Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis. Our analyses confirmed the presence of two distinct genetically divergent and geographically separated lineages of C. orientalis in Sri Lanka. The fast cost-effective gel-based PCR-RFLP marker method developed by us was successful in diagnosing C. orientalis from its closely resembling species. Thus, we believe our findings on the cryptic diversity and diagnostic methods will have important implications for the conservation and management of this endemic species.

Interaction of tobacco chewing and smoking habit with interleukin 6 promoter polymorphism in oral precancerous lesions and oral cancer.

Our study aims to determine the influence of smoking or tobacco chewing and the association of Interleukin 6 (IL-6) polymorphism, where G is substituted by A at the position - 596 (IL-6 - 596 G/A) and substitution of G by cytosine (C) at position - 572 (IL-6 - 572 G/C) on the susceptibility of precancerous oral lesions and oral cancer. The participants consisted of 250 subjects among which 75 were suffering from oral cancer, 75 subjects with precancerous oral lesions and 100 were healthy controls. Single-nucleotide polymorphism study (SNP) was done by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). IL-6 - 596 G/A SNP revealed genotypes, GG, and GA in subjects with precancerous oral lesions and oral cancer, and AA genotype was not found in any subject. IL-6 - 596 G/A was strongly associated with oral precancerous lesions but not with oral cancer. The present study reports that smokers carrying GA for IL-6 - 596 G/A were at several folds higher risk of developing oral precancerous lesions. Smokers with GC and CC for IL-6 - 572 G/C were at higher risk of developing oral precancerous lesions. No significant interaction was observed between these habits and IL-6 - 596 G/A and IL-6 - 572 G/C SNP with oral cancer. The interaction of variant A allele of IL-6 - 596 G/A and C allele of IL-6 - 572 G/C polymorphism with smoking and increases the risk of oral precancerous lesions. Tobacco chewing was not related with IL-6 - 596 G/A or IL-6 - 572 G/C in oral precancerous lesions or oral cancer. The study will help to determine the susceptibility of individuals with smoking or chewing habits to the development of oral precancerous lesion and oral cancer by monitoring the IL-6 SNPs which can be used as a biomarker for risk determination.