Reaction-restriction Fragment Length Polymorphism Procedure Research Articles

The point mutation analysis of Cyp2C9*2 (Arg144Cys C>T), Cyp2C9*3 (Ile359Leu A>C) and VKORC1 (1639G>A) in women with cervical cancer related to HPV: A case-control study.

Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide. HPV tumorigenesis genotypes are the causative agents of cervical cancer and genital malignancies. The scientific literaturehas demonstrated that life style, environmental, epigenetic accompanied with HR-HPV genotypes arepotential risk factors forcervical cancer progression. The frequencies of the Cyp2C9*2 , Cyp2C9*3 , and vitamin K epoxide reductase complex subunit 1 (VKORC1) genotypes as potential molecular biomarkers have been investigated on Iranian women with cervical malignancy related to HPV genotypes. As a case-control study, the mutations wereappraised using a polymerase chain reaction-restriction fragment length polymorphism procedure on women sufferingfrom HPV infection (60 cases), CC (46 cases), and 40 subjects of as healthy control. The outcomes demonstrated that Cyp2C9*3 showed a meaningful relationship between women diagnosed with cervical cancer and the healthy population (AA vs. AC; OR, 7.15; 95% CI, 1.94-26.3; p = .003). It was also observed that the Cyp2C9*3 mutation in women with cervical cancer and VKORC1 in healthy population with HPV (+), did not follow the Hardy-Weinberg equilibrium. Our findings aidunderstanding the genetic polymorphism distribution of Cyp2C9*2 , Cyp2C9*3 , and VKORC1 in women with genital malignancies. Thiscan also be useful in predicting the susceptibility risk factors for developing cervical cancer. However, allelic discrimination as a molecular biomarker requires further research.

The rs2167270 polymorphism of leptin gene is associated with atopic dermatitis.

ABSTRACTAtopic dermatitis is a chronic inflammatory skin disease that arises because of complex environmental, immunological, and genetic interactions. Adipokines are bioactive mediators secreted from adipocytes of white adipose tissue and are known to have a role in body metabolism and regulation of immune responses. Leptin is a proinflammatory adipokine that functions mainly to regulate food intake and energy expenditure. Few studies have implicated adipokines in the pathogenesis of atopic dermatitis. In this study, we investigated the association of three leptin gene polymorphisms: −2548G>A (rs7799039), −188 C/A (rs791620), and A19G (rs2167270), with the incidence of atopic dermatitis. One hundred and sixty-four patients and one hundred and sixty-seven age- and gender-matched controls were genotyped using the polymerase chain reaction-restriction fragment length polymorphism procedure. A significant association was found between rs2167270 and the incidence of atopic dermatitis (P < 0.05). The GG allele was more prevalent in the patients' group with genotype frequency of 38.7%, compared to 26.1% for the control group. No significant differences were found in the genotype distribution or allelic frequency of the other two examined polymorphisms, rs7799039 and rs791620, between atopic dermatitis patients and controls (P > 0.05). The results suggest that rs2167270 might play a role in the pathogenesis of atopic dermatitis.

Variants of NLRP3 gene are associated with insulin resistance in Chinese Han population with type-2 diabetes

AimsNod like receptor pyrin domain containing 3 (NLRP3) is the best characterized member of nod like receptor family. Recent studies suggest that NLRP3 plays a crucial role in the pathogenesis of type-2 diabetes (T2DM), and variants in NLRP3 affect its mRNA stability and expression. Therefore, we hypothesize that the variants in NLRP3 gene may contribute to T2DM susceptibility. The aim of this study is to evaluate the association of NLRP3 SNPs with T2DM in Chinese Han patients. MethodsTwo common variants in NLRP3 gene, rs10754558 and rs4612666, were detected using the polymerase chain reaction–restriction fragment length polymorphism procedure in 952 unrelated T2DM patients and 871 healthy controls. All participants were unrelated Chinese Hans. ResultsThe GG genotype and G allele frequencies of rs10754558 were significantly higher in T2DM patients than those in controls (for GG genotype, 19.6% vs. 14.5%, p=0.019; for G allele, 43.9% vs. 39.8%, p=0.013). The GG genotype of rs10754558 was significantly associated with higher LDL-C levels and more prone to insulin resistance, as evaluated by HOMA-IR or QUICK indexes. ConclusionsThe variant (rs10754558) in NLRP3 is related to insulin resistance and increased risk of T2DM in Chinese Han population.

Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines

Giardia duodenalis is a flagellated protist that causes gastrointestinal disease throughout the world. In the Philippines, study on G. duodenalis is limited. It is also believed that prevalence rates of this organism in the country are underestimated. In this study, stool samples from residents living in a slum area in Manila were collected. These were examined under microscopy for identification of common helminthic and protistan parasites. Results showed that 22.05% of 2,354 stool samples collected contained Giardia cysts. A fraction of samples (n = 133) positive for Giardia cysts were set aside. Genomic DNA was extracted from these samples and a polymerase chain reaction-restriction fragment length polymorphism procedure based on the organism's triose phosphate isomerase gene was utilized. This particular procedure is capable of distinguishing assemblages or genotypes within G. duodenalis. The highest identified assemblage was Assemblage B (86.47%). The two genotypes of Assemblage A were also detected. This is the first report on the identification of genotypes of G. duodenalis in the Philippines. The results of this study can serve as basis for future control and prevention of giardiasis and parasitism in the country.

Molecular taxonomy of theAlternariaandUlocladiumspecies from humans and their identification in the routine laboratory

The Alternaria and Ulocladium species reported from humans are studied taxonomically using rDNA internal transcribed spacer (ITS) sequence data. The ITS variability within the genus is relatively limited. The two most important, longicatenate species, Alternaria alternata and A. infectoria, clearly differ in their ITS domains, due to a 26-bp insert in ITS1 of the latter species. A number of taxa inhabiting particular plant species, such as A. longipes on tobacco and A. mali on apple, but also the common saprobic species A. tenuissima cannot reliably be distinguished from A. alternata using this method. The large number of described noncatenate, obligatory plant pathogens are extremely rare as agents of human disease; clinical routine identification does not need to include these taxa. The predictivity of a simplified polymerase chain reaction-restriction fragment length polymorphism procedure of rDNA for the recognition of the relevant species or species aggregates is established in a randomized test. The method was found to be rapid and cost-effective. Its efficacy extended to the identification of sterile and meristematic Alternaria strains, some of them previously classified in genera such as Chmelia and Botryomyces. Microscopic morphology and some additional tests remain necessary to allow identification of the aggregates of potential etiological agents of human disease. About 14% of the sequences deposited in GenBank were found to be misidentified. Alternaria infectoria is one of the most common clinical Alternaria species, despite its low degree of melanization. The lack of pigmentation has frequently led to misidentification of such isolates.

Studies on the HLA Class-II Antigens of a Patient Presenting a Double Alloimmunization Following Posttransfusion Purpura

In the Caucasian population, platelet incompatibility within the HPA-1 (Pl(A1/A2)) and HPA-5 (Br(a/b)) alloantigen systems are the two most likely causes of post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia. However, the way in which HLA (class-II) antigens participate in alloantibody formation is unclear. The patient (M-J.G.) is a middle aged woman with two children who developed a severe PTP (< 2000 platelets/µ1) 8 days after receiving red cell concentrates during coronary bypass surgery. During treatment with intravenous gamma-globulin and corticosteroids, her platelet count peaked, fell again, and returned to normal over a period of several months. Western blotting and/or the monoclonal antibody specific-immobilization of platelet antigens (MAIPA) assay performed with serum prepared at the height of her initial thrombocytopenia revealed antibodies to both the Pl(A1) and the Br(a) alloantigens. This rare combination prompted us to study the expression of specific HLA class II antigens in the patient. HLA-DR and DQ typing was performed from genomic DNA by the recently developed polymerase chain reaction-restriction fragment length polymorphism procedure (PCR-RFLP). The patient was found to express the DRB1*1302/1303 and DRB3*0101/0301 alleles (serological specificities: HLA-DR6 and DR52a/c respectively). She also expressed the DQA1*0102/0501, DQB1*0601 and DQB1*0301 alleles. Thus, this case provides further evidence linking an immune response to Pl(A1) and Br(a) antigens with HLA-DR52a/c and DR6.

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.