Restriction Fragment Length Polymorphism Assay Research Articles

3'-UTR Polymorphisms of MTHFR and TS Associated with Osteoporotic Vertebral Compression Fracture Susceptibility in Postmenopausal Women.

Postmenopausal osteoporosis is one of the most prominent diseases in postmenopausal women and it is increasing in prevalence with the aging population. Furthermore, osteoporosis and osteoporotic vertebral compression fractures (OVCFs) are related to mortality and decreased quality of life. Therefore, searching for biomarkers that are able to identify postmenopausal women who are at high risk of developing OVCFs is an effective strategy for improving the quality of life of patients and alleviating social and economic burdens. In this study, we investigated methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) gene polymorphisms in postmenopausal women with OVCF. We recruited 301 postmenopausal women and performed genotyping for the presence of MTHFR 2572C>A, 4869C>G and TS 1100C>T, 1170A>G. Genotyping was analyzed using the polymerization chain reaction restriction fragment length polymorphism assay. MTHFR 2572C>A and TS 1100C>T were associated with the prevalence of osteoporosis (MTHFR 2572CC versus CA+AA: odd ratio [OR] adjusted age, hypertention [HTN], and diabetes mellitus [DM] = 0.49, p = 0.012) and the occurrence of OVCFs (MTHFR 2572CC versus CA+AA: OR adjusted age, HTN, and DM = 0.38, p = 0.013; TS 1100CC versus CT+TT: OR adjusted age, HTN, and DM = 0.46, p = 0.02). Our novel finding is the identification of MTHFR and TS genetic variants that decrease susceptibility to OVCFs. Our findings suggest that polymorphisms in the MTHFR and TS genes are associated with susceptibility to osteoporosis and OVCFs in postmenopausal women.

Helicobacter Pylori Serology in Relation to Hepatitis C Virus Infection and IL28B Single Nucleotide Polymorphism.

The aim of the study was to evaluate the serological rate of Helicobacter pylori (H. pylori) infection in patients with chronic hepatitis C virus (HCV) infection and determine any correlations with liver damage and IL28B single-nucleotide polymorphism (SNP). One hundred eighty-nine patients with chronic HCV infection were included in the study, and H. pylori status was defined based on anti-H. pylori-IgG or anti-CagA-IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Liver damage was assessed using histology or transient elastography. IL28B C/T polymorphism (rs12979860) was evaluated in circulating blood cells using a PCR-based restriction fragment length polymorphism assay. Overall H. pylori serology was positive in 38.1% of our HCV-infected subjects. Among those, the anti-CagA-IgG positivity rate was 43.1% and was within the range of previously described populations of the same region. Highest prevalence of H. pylori was found in patients between 31 and 40 years compared to other age subgroups. The seropositivity rate was higher in the non-cirrhotic group than the cirrhotic one (45.4% vs. 20.0%, p < 0.05). No difference was found in IL28B genotype between H. pylori-positive and -negative cohorts. However, we observed a trend for the lower anti-CagA-IgG expression level in relation to the IL28B T-allele. Our results do not support an association between HCV and H. pylori infection. Whether IL28B SNP has a functional role in modulation of serological response to H. pylori CagA needs further investigation.

Genetic polymorphisms and protein expression of P53 and BRCA1 in preneoplastic and neoplastic rat mammary glands.

Breast cancer is the most common type of cancer and the leading cause of cancer-related deaths among women in the United Arab Emirates and worldwide. Although many factors contribute to the high incidence of breast cancer, a considerable number of cases are related to environmental factors. In the present study, breast cancer was induced in female rats using a single dose, 80mg/kg bodywt, of the environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The aim of the present study, was to characterize some of the molecular changes that occur during breast cancer development in the DMBA-treated rat model. Mammary gland tissues of control and DMBA-treated rats were processed for: i)immunohistochemical probing using anti-BRCA1 antibody to characterize and correlate the localization of this cellcycle protein during progression to cancer, ii)western blotting to analyze the alteration of p53 protein expression in preneoplastic and neoplastic lesions of the mammary glands, and iii)polymerase chain reactions using primers specific for BRCA1 and P53 genes followed by single stranded conformational polymorphism (SSCP) or restriction fragment length polymorphism (RFLP) assays to detect possible mutations in these genes during development of breast cancer. Microscopic examination revealed a wide range of preneoplastic and neoplastic lesions providing a sequence representing the multistep process of breast cancer formation in DMBA-treated rats. Probing for BRCA1 protein revealed a gradual defect in its translocation from the cytoplasm to the nucleus during breast cancer progression. In control rats, BRCA1 was present in the nuclei of terminal duct epithelial cells. However, in the preneoplastic lesions, BRCA1 was localized in both the cytoplasm and nuclei of the epithelial duct cells. In all malignant lesions, BRCA1 was mostly found in the cytoplasm. Western blotting revealed initial downregulation in the expression of p53 protein during breast cancer development. However, with progression towards malignancy, upregulation of p53 was observed. These changes were associated with polymorphism in p53 gene, which was detected in exon5 using SSCP assay. However, using RFLP and BamHI to digest the PCR products of exon11 of BRCA1 gene revealed no detectable polymorphisms. In conclusion, molecular characterization of the early changes that occur during development of breast cancer provides some clues for better understanding of its pathogenesis.

Molecular Characterization of Hepatitis D Virus Genotypes Circulating in Iran

Aim: To determine the molecular epidemiology and characterization of hepatitis D virus (HDV) genotypes circulating in different provinces of Iran. Patients &amp; methods: In this study, the presence of HDV RNA was tested in sera that were positive for hepatitis B surface antigen and HDV antibody by nested-PCR. HDV genotypes were subsequently analyzed using restriction fragment length polymorphism (RFLP) assay and then confirmed by sequencing. Results: 86.5% of positive PCR patients had genotype I and 8.1% had genotype II while the genotype of 5.4% of the patients remained undetermined by RFLP. Sequencing followed by phylogenetic analysis demonstrated that all the Iranian isolates were from genotype I. Conclusion: Although analyzing the RFLP of RT-PCR is a simpler method, the gold standard of genotyping of HDV is the phylogenetic analysis based on sequencing.

Association of P53 gene polymorphism with gastric cancer in Northern Iran as a high-risk region.

Gastric cancer has the fourth highest morbidity rate of all cancers worldwide. Genetic factors including alterations in oncogenes and tumor suppressor genes serve an important role in gastric cancer development and progression. The P53 gene acts as a tumor suppressor gene by regulating the cell cycle, DNA transcription and repair, apoptosis, senescence and genome stability. In addition to somatic P53 mutations in cancer development, germline polymorphisms are also involved in different malignancies. The polymorphism of P53 at codon 72 (Arg72Pro) is established as a common variant that increases susceptibility to various cancers. The present case-control study was conducted to evaluate the possible association between this P53 polymorphism and gastric cancer in the Iranian population. A total of 59 patients with gastric cancer and 59 healthy controls were enrolled in the present study. Genomic DNA was extracted from peripheral blood mononuclear cells and genotype analysis was performed using a polymerase chain reaction-based restriction fragment length polymorphism assay. Genotype frequencies did not differ significantly between the patients and controls (P=0.4); the frequencies of the three genotypes Arg/Arg, Arg/Pro and Pro/Pro in gastric cancer patients were 28.8, 49.2 and 22.0%, and in controls were 37.3, 49.2 and 13.6%. Additionally, there were no differences in genotype frequencies based on tumor location, histological differentiation or tumor stage. Based on these findings, it may be concluded that the P53 codon 72 polymorphism does not contribute to gastric cancer susceptibility in Northern Iran.

Identification of pathogenic mutations in two Chinese families affected with primary localized cutaneous amyloidosis

OBJECTIVE To identify potential mutations in two Chinese families affected with primary localized cutaneous amyloidosis. METHODS Peripheral blood samples of the family were collected with informed consent. Genomic DNA was extracted with a phenol chloroform method. All of the 17 exons and their flanking splicing sites of the OSMR gene were amplified with PCR and subjected to Sanger sequencing. Suspected mutations were verified with PCR - restriction fragment length polymorphism and high-resolution melting assays. RESULTS A missense mutation (c.1538G>A) was found in exon 10 of the OSMR gene in all of the six patients from family 1. A missense mutation (c.2081C>T) was found in exon 14 of the OSMR gene in all of the four patients from family 2. The same mutations were not found among the healthy controls. CONCLUSION Two missense mutations (c.1538G>A and c.2081C>T) were detected in the OSMR gene in two Chinese families affected with primary localized cutaneous amyloidosis. Our findings have further confirmed the pathogenicity of such mutations.

Identification, differentiation and antibiotic susceptibility of Gallibacterium isolates from diseased poultry

BackgroundGallibacterium anatis is an opportunistic pathogen of intensively reared poultry causing oophoritis, salpingitis, peritonitis and enteritis. Gallibacterium anatis infection often remains undiagnosed. Recently multi-drug resistant isolates have been described.MethodsA newly developed PCR restriction fragment length polymorphism assay targeting the 16S rRNA gene was used to identify and differentiate Gallibacterium isolates from chicken, turkey and partridge samples originating from 18 different geographical locations in Thuringia, Germany. Antimicrobial susceptibility to 19 compounds of different classes was assessed.ResultsNineteen Gallibacterium isolates were investigated. In 9 birds (47.4%) Gallibacterium species were isolated exclusively while in 10 birds (52.6%) other bacterial or viral agents could be detected in addition. In one chicken a mixed infection of Gallibacterium anatis and Gallibacterium genomospecies was identified. All isolates were susceptible to apramycin, florfenicol and neomycin and resistant to clindamycin, sulfathiazole and penicillin. Resistance to sulfamethoxim, spectinomycin, tylosin and oxytetracycline was observed in 93.3%, 93.3%, 86.7% and 80.0% of the field strains, respectively.ConclusionsThe PCR-RFLP assay allows specific detection and differentiation of Gallibacterium spp. from poultry. Antimicrobial resistance of Gallibacterium spp. is highly significant in Thuringian field isolates.

Isolation of Sabin-like Polioviruses from Sewage in Poland.

As a complement to the active search for cases of acute flaccid paralysis, environmental sampling was conducted from January to December 2011, to test for any putative polio revertants and recombinants in sewage. A total of 165 environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture (L20B, RD and Caco-2) followed by neutralization and reverse-transcription polymerase chain reaction. Out of the 31 CPE positive samples, 26 contained one and 5 two different serotypes, yielding a total of 36 PVs. The microneutralization test revealed the presence of 7, 10 and 19 strains belonging to poliovirus serotype 1, 2 and 3, respectively. The genomic variability of 36 poliovirus strains was examined by the restriction fragment length polymorphism assay (RFLP). By combined analyses of two distant, polymorphic segments of the viral genome, one situated in the capsid protein VP1 coding region and the other in the 3D-polymerase coding region, we screened for the putative poliovirus revertants and recombinants. All detected PVs were classified as vaccine strains on the basis of RFLP-VP1 test. None of wild-type PVs or vaccine derived polioviruses were detected. RFLP assay also revealed the presence of 11 recombinants in 3D-polymerase coding region. Nine isolates appeared to be S3/S2, one S3/S1 and S1/S2 recombinant in analyzed 3Dpol region. This study revealed, through environmental monitoring, the introduction of SL PVs into the population associated with the routine use of OPV in Poland before the April 2016. Our findings demonstrate the usefulness of environmental surveillance in the overall polio eradication program.

Association of C3435T, C1236T and C4125A Polymorphisms of the MDR-1 Gene in Egyptian Children with Acute Lymphoblastic Leukaemia

Background:P-glycoprotein (P-gp), a membrane transporter encoded by the multidrug resistance-1 (MDR1) gene, influences pharmacokinetics and metabolism of anticancer drugs and contributes to multidrug resistance phenotype in acute lymphoblastic leukemia (ALL). Genetic variation ofMDR1 in ALL patients is increasingly recognized as a factor influencing response to treatment.Aim:To investigate the possible role of MDR-1 gene polymorphisms (C3435T, C1236T and C4125A) as risk factors for the development and clinical outcome of ALL in Egyptian children.Materials and Methods:Genotyping of MDR-1 C3435T, C1236T and C4125A single nucleotide polymorphisms (SNPs) was accomplished using a polymerase chain reaction–restriction fragment length polymorphism (RFLP-PCR) assay with 120 childhood ALL patients and 100 healthy controls.Results:Homozygous T with the C3435T SNP showed a protective effect as compared to homozygous C (OR=0.748) while heterozygous CT correlated with a poor outcome (high risk, drug unresponsiveness, relapse and high percentage of death). Additionally, the T allele of the C1236T SNP showed a significant relation with ALL risk (OR=1.6). However, there were no significant differences in the genotype and allele frequencies of MDR-1 SNPs between patients and controls. Only one genotype (CC) and one allele of MDR-1 (C4125A) were seen. Neither CA/AA genotypes nor A alleles were present in ALL patients and normal controls. TC was the predominant haplotype in both groups, while CT proved to be minor. The cumulative incidence of relapse was higher with the CC genotype of C1236T as compared with TT.Conclusion:From our preliminary data, the CT genotype of C3435T is associated with a poor ALL outcome while the CC genotype of C1236T is related with an increased incidence of relapse. Although our results provide assistance for oncologist choice of individual therapeutic strategy taking the patient genetic repertoire into consideration, further investigations with larger sample size should be conducted to validate our results.

Association of the common SNPs in RNF212, STAG3 and RFX2 gene with male infertility with azoospermia in Chinese population

ObjectiveThe aim of this study was to explore the association between the SNP rs4045481 in RNF212 gene, rs1050482 and rs11531577 in STAG3 gene as well as rs2288846 in RFX2 gene and male infertility with azoospermia in Chinese population. Study designTwo hundreds and twenty infertile patients with azoospermia and 248 fertile men were recruited in the present study. The four SNPs investigated were genotyped using polymerase chain reaction and restriction fragment length polymorphism assay. The differences in allelic and genotypic frequencies between patients and controls were evaluated by chi-square test. ResultsNo significant differences in allele and genotype frequencies of SNP rs1050482 and rs11531577 in STAG3 gene as well as rs2288846 in RFX2 gene between patients with azoospermia and controls were observed. However, the frequencies of allele C(43.6% vs. 34.1%, P = 0.003, OR = 1.498, 95% CI 1.150–1.192) and genotype CC (24.6% vs. 12.0%, P = 0.001, OR = 2.346, 95% CI 1.448–3.858) were significantly higher in patients with azoospermia than those in controls at the rs4045481 locus in RNF212 gene. ConculusionThe polymorphism of SNP rs4045481 in RNF212 gene might be associated with azoospermia and genotype CC of this SNP may be a risk factor of azoospermia.

TNF-α-308 polymorphism determines clinical manifestations and therapeutic response of ankylosing spondylitis in Han Chinese

Background and objectiveThere is ongoing debate as to whether tumor necrosis factor alpha (TNF-α)-308 is associated with ankylosing spondylitis (AS). The aim of the present study was to determine whether TNF-α-308 is involved into genetic susceptibility, clinical features and therapeutic response of AS in Han Chinese. MethodsTwo hundred and sixty AS patients with 260 ethnically matched healthy blood donors were enrolled into the present study. TNF-α-308 promoter polymorphism was identified using polymerase chain reaction amplification with restriction fragment length polymorphism assay. ResultsPopulation genetic analysis showed that the prevalence of allele A and G/A genotype was equally infrequent in both AS patients (3.85% and 7.69%) and healthy subjects (4.23% and 8.46%). Compared with the carriers of G/G genotype, remarkably elevated erythrocyte sedimentation rate and serum C-reactive protein were observed in AS patients with G/A variant (87.06±49.40 vs. 55.53±42.99mm/h, p=0.0126; 54.95±27.77 vs. 34.36±36.13mg/dl, p=0.0116, respectively), and they always presented with inflammatory spinal pain (70.00% vs. 43.33%, p=0.0214) and suffered relatively mild sacroiliitis (65.00% vs. 41.67%, p=0.0431). The allele G and G/G genotype were more frequent in good responders to anti-TNF-α treatment (96.55% vs. 73.53%, p=0.0032; 93.10% vs. 47.06%, p=0.0015), whereas there was no obvious superiority of them in predicting therapeutic response of conventional medications for AS. ConclusionsOur data suggest that TNF-α-308 polymorphism may influence the clinical features rather than susceptibility to AS in our Han Chinese.

Effects of MPO-463G/A and -129G/A polymorphisms on coronary artery disease risk and patient survival in a Turkish population.

Myeloperoxidase (MPO) is an oxidative hemoprotein compound expressed in polymorphonuclear leukocytes that contributes to inflammatory responses. Coronary artery disease (CAD), as the most prevalent form of heart disease, is considered to originate from an interaction between genetic and environmental factors. In the present study, the potential associations between MPO-463G/A and -129G/A polymorphisms with CAD were investigated in a Turkish population using a polymerase chain reaction-based restriction fragment length polymorphism (RFLP) assay technique. To the best of our knowledge, the study was the first to examine the association of MPO-463G/A and -129G/A with patient survival rate in a Turkish population. The study population consisted of 201 patients with CAD and 201 healthy controls. The results indicated that there was a significant association of the GA genotype of MPO-463G/A with the case population (P=0.048). Meanwhile, in the patients with CAD, the frequency distributions of the MPO-129A allele (P=0.006) and GA genotype (P=0.001) were significantly increased compared with the G allele and GG genotype, respectively, in CAD patients. Additionally, compared with the GG genotype, the frequency distribution of MPO-129A was significantly increased in the patient group regarding smoking status (P=0.001) and the presence of hypercholesterolemia (P=0.028). However, survival analysis did not detect an effect of either polymorphism on the survival rate of the CAD patients (P>0.05). Therefore, the MPO-129GA genotype may be a significant risk factor for the development of CAD.

Synergistic effect of collagenase-1 (MMP1), stromelysin-1 (MMP3) and gelatinase-B (MMP9) gene polymorphisms in breast cancer.

BackgroundExtracellular matrix degradation by matrix metalloproteinases (MMPs) is an important mechanism involved in tumor invasion and metastasis. Genetic variations of MMPs have shown association with multiple cancers. The present study is focused to elucidate the association of MMP-1, 3 and 9 genetic variants with respect to epidemiological and clinicopathological variables by haplotype, LD, MDR, survival in silico analyses among South Indian women.Material and methodsMMP3–1171 5A/6A and MMP9–1562 C/T SNPs were genotyped by Allele specific polymerase chain reaction and MMP1-1607 1G/2G polymorphism by restriction fragment length polymorphism assays respectively, in 300 BC patients and age-matched 300 healthy controls. Statistical analysis was performed using the SNPStats and SPSS software. Linkage disequilibrium and gene-gene interactions were performed using Haploview and MDR software respectively. Further, transcription factor binding sites in the promoter regions of SNPs under study were carried out using AliBaba2.1 software.ResultsWe have observed an increased frequency of 2G-allele of MMP1, 6A-allele of MMP3 and T-allele of MMP9 (p<0.05) respectively in BC subjects. The 2G-6A haplotype (minor alleles of MMP-1 and MMP-3 respectively) has shown an increased susceptibility to BC. Further, MMP polymorphisms were associated with the clinical characteristics of BC patients such as steroid hormone receptor status, lymph node involvement and metastasis. SNP combinations were in perfect LD in controls. MDR analysis revealed a positive interaction between the SNPs. 5-years survival rate and cox-regression analysis showed a significant association with clinicopathological variables.ConclusionOur results suggest that MMP1–1607 1G/2G, MMP3–1171 5A/6A and MMP9–1562 C/T gene polymorphisms have synergistic effect on breast cancer. The interactions of MMPs clinical risk factors such as lymph node involvement has shown a strong correlation and might influence the 5-years survival rate, suggesting their potential role in the breast carcinogenesis.

Influence of the MIF polymorphism -173G>C on Turkish postmenopausal women with osteoporosis.

MIF, aproinflammatory cytokine, contributes to the pathogenesis of acute, chronic, and autoimmune inflammatory disorders and balances the suppressive effect of glucocorticoids on the immune system. There is an interaction between bone metabolism and the immune system via the production of cytokines. We aimed to analyze the relationship between the MIF gene -173G> C promoter polymorphism and osteoporosis. In this case-control study performed in auniversity hospital, 286 samples (136 women with osteoporosis and 150 healthy age-matched controls) participated. The polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was used to genotype the MIF gene polymorphism. The alleles and genotypes frequencies of patients and controls were compared using the χ2 test. The genotype frequencies of MIF gene -173G> C polymorphism showed statistically significant differences between patients and controls (p= 0.038). Also, the subjects carrying the variantC allele in the MIF -173 position were at significantly higher risk of osteoporosis than subjects carrying the wild-typeG allele (p= 0.009, odds ratio 1.7, 95% confidence interval 1.1-2.6). Our study suggested astrong association between MIF gene -173G> C polymorphism and osteoporosis in aTurkish population.

IL-1β haplotype influences the effect of NOx exposure on gestational age in the South African MACE birth cohort.

Cytokines, molecules within the immune system that affect either a pro- or anti-inflammatory response, have previously been shown to influence birth outcomes. The maternal cytokine gene-environment interactions are thought to alter their expression, potentially influencing susceptibility to adverse birth outcomes. The aim of this study was to determine the association between the maternal interleukin-1β (IL-1β) haplotype and expression variation with oxides of nitrogen (NOx) levels, and thereafter investigate the IL-1β haplotype-specific effects of NOx exposure levels, IL-1β mRNA expression and other variables on gestational age. Using the prospective Mother and Child in the Environment (MACE) birth cohort in Durban, South Africa, 335 participants were genotyped for the IL-1β haplotype. Previous studies showed that three single nucleotide polymorphisms (SNPs), IL-1β-1464G/C, -511C/T and -31C/T, constitute the IL-1β functional haplotype. These SNPs were genotyped using a restriction fragment length polymorphism assay, while IL-1β mRNA expression was measured using a quantitative real-time polymerase chain reaction assay. Individual estimates of NOx exposure were obtained by land use regression modelling. A multivariate linear regression analysis was employed to test for significant effects on gestational age. IL-1β mRNA expression was found to possess a haplotype-dependent effect ( p = 0.0001) and its expression levels positively correlated with NOx levels ( r = 0.34; p = 0.006). In the high haplotype model, a unit increase in NOx exposure level was associated with a decrease in gestational age by 1 week ( p = 0.02). Furthermore, gestational age decreased by 0.9 weeks for every unit increase of IL-1β mRNA expression level ( p = 0.025). HIV-1 positivity was associated with a 0.2-week decrease in gestational age ( p = 0.035) in the intermediate haplotype model and a 0.4-week decrease in the high haplotype model ( p = 0.044). These data have implications for better understanding the effect of prenatal NOx exposure on gestational age and demonstrate the role of the IL-1β haplotype in modulating the effects of NOx exposure.

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Investigation of XRCC1 Arg399Gln, Arg280His, and Arg194Trp polymorphisms effects on the induction of micronucleus by Aflatoxin B1 in in vitro

Aflatoxin B1 (AFB1) is a genotoxic mycotoxin that can contaminate a wide variety of foods. The aim of this study was to examine the associations between XRCC1 Arg399Gln, Arg280His, and Arg184Trp polymorphisms and the frequencies of spontaneous and AFB1-induced DNA damage in peripheral blood lymphocytes from 100 healthy individuals in Turkish population. In vitro cytokinesis-blocked micronucleus (CMBN) assay was used to detect the spontaneous and AFB1-induced DNA damage. Genotyping of XRCC1 Arg399Gln, Arg280His, and Arg194Trp polymorphisms were carried out by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. Treatment of the lymphocytes with AFB1 slightly increased (for Arg399Gln 0.73±0.4 vs. 0.40±0.26, Arg280His 0.80±0.4 vs. 0.41±0.28) the overall frequencies of micronucleus (MN) when compared to untreated cultures. No results were obtained for Arg194Trp polymorphism. Obtained overall results do not correspond to any significant (P<0.05) association between XRCC1 Arg399Gln, Arg280His, and Arg194Trp Polymorphisms and MN frequencies. The results of this study indicate that Arg399Gln, Arg280His, and Arg194Trp polymorphisms do not play an important role in human sensitivity to the genotoxic effects of AFB1.

A novel synonymous SNP (A47A) of the &amp;lt;i&amp;gt;TMEM95&amp;lt;/i&amp;gt; gene is significantly associated with the reproductive traits related to testis in male piglets

Abstract. Transmembrane protein 95 (TMEM95) is located on the acrosomal membrane of the sperm head involved in the acrosome reaction; thus, it is regarded as affecting spermatogenesis and reproduction traits. The aim of this study was to explore the novel single nucleotide polymorphisms (SNPs) within the pig TMEM95 gene as well as to evaluate their associations with the testicular sizes in male Landrace (LD) and Large White (LW) breeds. After pool sequencing and bioinformatics analysis, only one novel coding SNP was found in exon 1, namely NC_010454.3: g.341T &gt; C, resulting in a synonymous mutation (A47A). This SNP could be genotyped using the StuI polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. The minor allelic frequencies (MAFs) were 0.259 and 0.480 in the LD and LW breeds. Their polymorphism information content (PIC) values were 0.310 and 0.375. The LW population was at the Hardy–Weinberg equilibrium (HWE) (p &gt; 0.05), whereas the LD population was not (p &lt; 0.05). Association analyses demonstrated that a significant relationship was found between this A47A polymorphism and testis weight at 40 days of age in the LW population (p = 0.047), and the heterozygote individuals showed lower testis weight than those with other genotypes. Moreover, this SNP was significantly associated with three testis measurement traits at 15 days of age in the LW population (p &lt; 0.05); the individuals with genotypes TT and TC showed consistently superior testis measurement traits than those with genotype CC. These findings demonstrate that the A47A polymorphism had a significant effect on testis measurement traits, suggesting that the TMEM95 gene could be a candidate gene associated with reproductive traits. These results could contribute to breeding and genetics programs in the pig industry via DNA marker-assisted selection (MAS).

Rheumatoid arthritis is associated with rs17337023 polymorphism and increased serum level of the EGFR protein

ObjectiveWe have previously described the association of rheumatoid arthritis (RA) prevalence and two epidermal growth factor receptor (EGFR) SNPs (rs17337023 and rs2227983) among the Taiwanese population. This present study aimed to elucidate whether the SNPs can alter the expression of EGFR in the progression of RA.MethodsThe cohort study included 366 Taiwan’s Han Chinese RA patients and 326 age and gender matched healthy controls. Blood samples collected from the participants were analyzed to determine their serum EGFR levels and to identify EGFR SNPs from their genomic DNA. Genotyping for EGFR SNPs was performed by restriction fragment length polymorphism (RFLP) assay. The relationship between EGFR SNP and the clinical manifestations of RA was evaluated.ResultsOur results showed that a statistically significant difference in genotype frequency distributions at rs17337023 SNP for RA patients and controls (p ˂ 0.05). In addition, compared with the haplotype frequencies between case and control groups, the RA patient with the GT haplotype appeared to be a significant “protective” haplotype compared with other haplotypes (OR: 0.73, 95% CI: 0.59–0.91; p = 0.005). Furthermore, the increased serum level of EGFR was also observed in RA patients (p ˂ 0.001).ConclusionOur study showed that RA is associated with rs17337023 SNP in EGFR gene and increased serum level of the EGFR protein. These findings suggest EGFR is worthy of further investigation as a therapeutic target for RA.

Differential diagnosis between ts-11 vaccine strain and field Mycoplasma gallisepticum isolates in clinical samples by PCR-RFLP

Summary Mycoplasma gallisepticum ( MG) is an important avian pathogen causing significant economic losses within the poultry industry. The aim of the present study was to investigate the prevalence of MG in poultry by PCR of mgc2 and 16S rRNA and to set a differential diagnosis between vaccine strain and field MG. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the 16S rRNA and mgc2 genes present in MG. For the 16S rRNA and mgc2 PCR assays, 109 samples were taken from 10 RSAT positive farms, including: lung, air sacs and tracheal swabs. For differential diagnosis between the ts-11 vaccine strain and other field isolates, PCR-RFLP with HaeII restricted enzyme was applied. PCR products of 530Â bp and 300Â bp, respectively, appeared on electrophoresis gel with 16S rRNA and mgc2 PCR diagnostic primers specific for MG. Differential diagnosis of MG can be achieved within 1 day of submission of tracheal swab samples by application of the mgc2-PCR–restriction fragment length polymorphism (mgc2-PCR-RFLP) assay with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains. The test was successfully applied in vivo for detection of MG, and RFLP-PCR can be used for rapid differentiation of the ts-11 vaccine strain from field isolates by direct amplification from clinical samples without the need for isolation by culture.