Tetra-primer Amplification Refractory Mutation System-polymerase Chain Reaction Research Articles

PIK3CA rs7640662 (C/G) single nucleotide polymorphism lacks association with breast cancer cases in Persians.

Phosphatidylinositol-3-kinase (PI3K) is a group of enzymes involved in cellular growth, proliferation, differentiation, cell motility, intracellular trafficking, and survival that play very important roles in developing breast cancer. PIK3CA is a gene that encodes α catalytic subunit of this enzyme. A common polymorphism of PIK3CA, rs7640662 (C/G), was analyzed, and its association to breast cancer cases was determined. In this study, DNA was extracted from peripheral blood samples of 278 women suffering from breast cancer and 128 healthy women. Tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method was performed to genotype rs7640662. P values and ODD ratios were measured using SPSS. P value less than 0.05 and ODD ratios more than 1 were considered as significant. All ODD ratios were less than 1, and P values were more than 0.05 showing that rs7640662 (C/G) and breast cancer are not significantly associated. However, the genotypes observed in the Persian population, as an ancient population living in the Middle East, was significantly different from the genotypes reported by HapMap for Asian populations. As a conclusion, rs7640662 was not associated with the risk of breast cancer in a Persian population; however, it was observed that heterozygote (GC) is the most common genotypes in both case and control samples.

Association of polymorphisms -319 C/T and +49 A/G of CTLA-4 gene with Chronic Hepatitis C virus Infection in Mexican population

Association of polymorphisms -319 C/T and +49 A/G of CTLA-4 gene with Chronic Hepatitis C virus Infection in Mexican population

Investigation of the association between paraoxonase-1 gene polymorphisms and response to therapy in chronic hepatitis C patients

Liver-derived paraoxonase-1 (PON1) enzyme that is found in the circulation is bound to high-density lipoproteins and reduces the amount of oxidized lipids with its antioxidant effect. Humans have at least three different PON gene regions which are adjacent to the other on the 7th chromosome. It has been shown that PON1 gene and its polymorphisms are related with various diseases. It is also known that, hepatitis C virus (HCV) is tightly associated with the cell lipoproteins in each step of its replication cycle leading to modulation of the host lipid metabolism. The aim of this study was to investigate the relationship between the response to chronic hepatitis C (CHC) therapy and aminoacid changes in 55' and 192' regions of PON1 enzyme believed to be involved in the pathophysiology of many chronic diseases. A total of 49 CHC patients (27 male, 22 female; mean age: 52.9 ± 12.6 yrs), all infected with HCV genotype 1b and positive for anti-HCV and HCV-RNA were included in the study. Patients who were HCV-RNA negative at the sixth month following at least once pegilated interferon + ribavirin treatment, were considered as therapy-responders, whereas those who were HCV-RNA positive were considered as non-responders. The genomic DNAs were isolated from patients' blood samples in their routine follow-ups and Q/R192 and L/M55 PON1 polymorphism analysis in 55. and 192. regions was performed by T-ARMS-PCR (Tetra-primer amplification refractory mutation system-polymerase chain reaction) method. In our study, the analysis of PON1 polymorphisms yielded 44.1% of LL, 44.1% of LM and 11.8% of MM genotypes at position 55 and 55.9% of QQ, 41.2% of QR, and 2.9% of RR genotypes at position 192 in therapy-responders. In the evaluation of combined genotype analysis of the patients, there was only one case who was responsive to treatment with LL/RR genotype. Of the patients, eight harbored LL/QQ genotypes and seven of them (87.5%) were responsive to treatment. However, statistical analysis indicated that there was no relationship between PON1 L/M55 and PON Q/R192 polymorphisms and response to CHC treatment (chi-square test, p> 0.05). Our data did not support a relationship between PON1 polymorphisms and response to CHC therapy, in contrast to a few studies pointing out of this correlation. This might be attributed to relatively low number of patients included. In conclusion, since antiviral agents used for CHC therapy are limited and costly, it was thought that further investigations with large numbers of patients should be conducted to establish the presence of any relationship between the response to CHC therapy and genotypes of the PON1 enzyme.

A Multiplex Polymerase Chain Reaction Method for the Simultaneous Detection of GSTM1, GSTT1, and GSTP1 Polymorphisms

Glutathione S-transferases (GSTs) play an important role in the detoxification of a wide variety of toxicants. Among GSTs, GSTM1 and GSTT1 polymorphic deletions and the GSTP1 Ile105Val polymorphism were often studied in combination in many diseases because of their additive effects, but they were usually genotyped by two separate methods. The purpose of the present study was to develop a simple and reliable method to simultaneously detect these three polymorphisms. The three polymorphisms of 259 volunteers were genotyped using a multiplex polymerase chain reaction (PCR) method based on a tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR), and the results were validated by multiplex PCR for GSTM1 and GSTT1 polymorphisms and DNA sequencing of the GSTP1 Ile105Val polymorphism, respectively. The multiplex PCR method of GSTM1, GSTT1, and GSTP1 polymorphisms based on T-ARMS-PCR can simultaneous detect the three polymorphisms in a single PCR. The results of this method are in perfect accord with the results of the multiplex PCR method of determining GSTM1, GSTT1 polymorphisms, and DNA sequencing of GSTP1 Ile105Val polymorphism. The novel multiplex PCR method of GSTM1, GSTT1, and GSTP1 polymorphisms is simple, fast, low-cost, and reliable for the simultaneous detection of GSTM1, GSTT1, and GSTP1 Ile105Val polymorphisms.

Quantitative Analysis of Interdental Gingiva in Patients With Chronic Periodontitis and Transforming Growth Factor‐β1 29C/T Gene Polymorphisms

The association of transforming growth factor (TGF)-β1 29C/T gene polymorphisms with level of tissue breakdown and periodontal disease progression is not clear. In this study, quantitative parameters of interdental papilla are investigated in patients with chronic periodontitis (CP) and TGF-β1 29C/T gene polymorphisms. Sixty gingiva samples were included. After determination of TGF-β1 29C/T gene polymorphisms using tetra-primer amplification refractory mutation system/polymerase chain reaction (T-ARMS-PCR), 15 gingival tissue samples from patients with CP in each genotype (TT, TC, and CC) were considered as case groups. Fifteen control samples were also collected from healthy individuals. After tissue processing, interdental gingiva tissues were exhaustively sectioned into 4-μm-thick sections. Ten to 13 sections were sampled by systematic uniform random sampling and stained with Masson trichrome, and the volume density (Vv) of the gingival components was estimated using Cavalieri estimation. Statistically significant differences were found in Vv of epithelium, connective tissue, collagenous and non-collagenous matrix, and blood vessels between control and CP groups (P <0.0001). There was a corresponding decrease in the collagenous matrix Vv in patients with the TT genotype compared with those with CT and CC genotypes. Collagenous matrix and blood vessel Vv values were statistically correlated with the number of T alleles (r = -0.74, r(2) = 54.8%, P = 0.0001 and r = 0.84, r(2) = 70.6%, P = 0.0001, respectively). This study shows that there is a strong association between TGF-β1 29C/T gene polymorphisms and quantitative parameters of interdental papilla in patients with CP.

Genotyping the CRHR1 rs242939 (A&gt;G) Polymorphism by a One-Step Tetra Primer–Amplification Refractory Mutation System–Polymerase Chain Reaction

With the rapid advances in molecular techniques, various methods for genotyping single-nucleotide polymorphisms (SNPs) are available. Still, the search for easy, robust, and less costly techniques continues. We wished to develop a Tetra primer-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) based technique for the corticotrophin-releasing hormone receptor 1 (CRHR1) (rs242939) SNP for use in our research lab. To detect SNPs in a single-step PCR, we set up two genotyping methods, T-ARMS-PCR and restriction fragment length polymorphism (RFLP). The study was performed using thirty blood samples taken from clinically defined asthmatic patients. The efficiency and effectiveness of results obtained by T-ARMS were validated by PCR-RFLP and sequencing. This study demonstrates that T-ARMS is feasible and applicable to discriminate a wild-type allele from the respective mutant allele in one step. This work is the first that presents a rapid, sensitive, and high throughput genotyping method for the CRHR1 (rs242939) polymorphism and can be used for both large- and small-scale genotyping studies.

A Tetra-Primer Amplification Refractory Mutation System–Polymerase Chain Reaction for the Detection of rs8099917 IL28B Genotype

Two single nucleotide polymorphisms (SNP, rs8099917, rs12979860) near the IL28B gene have been illustrated as outcome predictors of hepatitis C virus (HCV) treatment with pegylated interferon/ribavirin. The aim of the present study was to design a simple tetra-primer amplification refractory mutation system–polymerase chain reaction (T-ARMS-PCR) for the evaluation of the rs8099917 IL28B gene polymorphism. We efficiently designed a T-ARMS-PCR for detection of rs8099917 IL28B. Using this method, we genotyped 83 subjects with NAFLD (nonalcoholic fatty liver disease) and 93 healthy subjects. No significant differences were found in allelic and genotypic frequencies of rs8099917 IL28B gene polymorphism between NAFLD subjects and controls. The frequency of minor allele (G allele) was 0.13 in healthy and 0.19 in NAFLD subjects. In conclusion, we designed a simple, inexpensive, and reproducible T-ARMS-PCR for detection of rs8099917 IL28B polymorphism which can be used for routine assay. In addition, we found that rs8099917 polymorphism is not a risk factor for predisposition to NAFLD in a sample of Iranian population. Larger studies with different ethnics are required to validate our findings.

A tetra-primer amplification refractory mutation system polymerase chain reaction for the evaluation of rs12979860 IL28B genotype

Recently, genome-wide association studies in patients affected by HCV infection have identified a strong association between sustained virological response to peg-interferon/ribavirin and spontaneous viral clearance and common single nucleotide polymorphisms (SNPs) near the IL28B gene, encoding for interferon-lambda-3. Thus, it is anticipated that IL28B genotype determination will be integrated in clinical practice to guide treatment decisions. Here, we describe a simple tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) for the evaluation of the rs12979860 C>T IL28B SNP, for which strong evidence of association with clinical outcomes has been collected in subjects of European descent. Valid genotypic data were obtained for over 99% of subjects analysed, and T-ARMS-PCR procedures were validated by the analysis of DNA samples of 164 patients with chronic HCV infection. In conclusion, this method allows rapid, reproducible, inexpensive and accurate detection of rs12979860 polymorphism without need of any special equipment and is also suitable for evaluation of a low number of samples on a routine basis.

Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR

The G-->A transition at nucleotide 21881 of the human catechol-O-methyltransferase (COMT) gene represents a functional genetic polymorphism (Val158Met), rendering an enzyme with reduced activity that has been associated with psychiatric disorders and estrogen-related cancers. A new method for the detection of this polymorphism is described, based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), with a single PCR to discriminate both alleles. Two primers amplify a common amplicon independently of the allele considered. At the same time, two primers are used, differing in the 3' base. In the Val/Val or Met/Met conditions, amplification occurs both in the general amplicon and in the specific allele; in the Val/Met condition three different amplicons are produced. Direct DNA sequencing of a COMT region containing the G/A polymorphism demonstrates the validity of this tetra-primer ARMS-PCR method. Reevaluation by PCR-RFLP revealed 100% accordance for genotype adscription. Subjects carrying the COMT(HH) genotype in a Spanish population comprised 28%, and the COMT(LL) homozygotes amounted to 21%. The described method provides a fast and reliable approach for determining COMT polymorphism that can be useful in large clinical studies using minimal quantity of DNA, avoiding the timely and costly use of restriction enzymes.