Amplification Refractory Mutation System-polymerase Chain Research Articles

Development and application of low-cost T-ARMS-PCR assay for AGT and CYP11B1 gene polymorphisms.

Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-β-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-β-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-β-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.

Applicable Value of AMSS-PCR in Lung Cancer Gene Mutation Detection

背景与目的肺癌驱动基因检测具有重要意义，目前检测方法多样，临床适用性有差异。本研究旨在比较基于扩增阻碍突变系统-聚合酶链反应(Amplification Refractory Mutation System-polymerase chain reaction, ARMS-PCR)技术的试剂盒与一代测序及ARMS-qPCR检测肺癌突变基因的敏感性和特异性，探究突变位点特异扩增法(Amplification Mutation Specific System, AMSS)-PCR技术在肺癌突变基因检测中的应用价值。方法对前期已行ARMS-PCR检测的肿瘤标本进行一代测序及试剂盒检测，比较各种方法的检测结果，并对检测结果进行统计学分析。结果本研究共收集了309例肺癌标本。试剂盒与一代测序符合率97.41%，ARMS-PCR的符合率97.73%。试剂盒与一代测序、试剂盒与实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, qPCR)、qPCR与一代测序一致性检验的Kappa值分别为0.946、0.953、0.913。试剂盒以一代测序为参照的受试者工作特征曲线(receiver operating characteristic curve, ROC)曲线下面积为0.976，以qPCR为参照的ROC曲线下面积为0.975。结论AMSS-qPCR技术能够有效检测肺癌突变基因，具有较好的临床应用价值。

Angiotensin II type 1 receptor gene polymorphism and serum angiotensin-converting enzyme level in Egyptian children with systemic lupus erythematosus.

Angiotensin II, the major effective molecule of the renin-angiotensin system, plays a vital role in the development of systemic lupus erythematosus (SLE). To study angiotensin II type 1 receptor (AT1R) gene polymorphism at (A1166C) in Egyptian children with SLE and its correlation with serum ACE level and SLE manifestations. AT1R gene polymorphism (A1166C) was done in 123 children with SLE in comparison to 100 healthy controls using polymerase chain reaction-based restriction fragment length polymorphism method (PCR-RFLP) and the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) to confirm the results of the genotyping. Serum ACE level measurement was done using ELISA technique. The frequencies of C-containing genotypes (AC + CC) and C-allele of AT1R (A1166C) were significantly higher in SLE patients compared to controls (p < 0.0001, OR = 4.9, 95% CI = 2.7-8.8; p˂0.0001, OR = 3.6, 95% CI = 2.2-5.9, respectively). Lupus nephritis (LN) patients had significantly higher frequency of (AC + CC) genotypes and C-allele compared with controls (p˂0.0001, OR = 5.1, 95% CI = 2.7-9.7; p˂0.0001, OR = 3.5, 95% CI = 2.1-6.02, respectively). Mean serum ACE levels were significantly higher in SLE patients compared to controls (p˂0.0001). There were no associations between AT1R gene polymorphism and serum ACE level and the clinical manifestations of SLE. The AT1R gene polymorphism can be considered a risk factor for the development of SLE in Egyptian children.

P2X7 receptor polymorphisms and susceptibility to tuberculosis in a North Indian Punjabi population

Genetic elements are known to influence susceptibility to tuberculosis (TB). P2X7R is a candidate gene with multiple single-nucleotide polymorphisms (SNPs) that has the potential to influence an individual's ability to kill the intracellular pathogen Mycobacterium tuberculosis. To explore the role of five functional polymorphisms of P2X7R in susceptibility or resistance to TB in a North Indian Punjabi population. A case-control study was conducted among 245 TB patients (145 pulmonary TB [PTB] and 100 extra-pulmonary TB [EPTB]) and 247 healthy controls. DNA extracted from samples of peripheral blood was analysed for five SNPs of P2X7R using amplification refractory mutation system-polymerase chain reaction (PCR) [-762(T/C), +1513(A/C), DNA sequencing +1729(T/A)] and PCR-restriction fragment length polymorphism [+489(G/A), +946(G/A)] methods. Of the three loss-in-function polymorphisms, +1513(A/C) showed a statistically significant association with TB susceptibility, while the other two (+946 and +1729) sites were found to be monomorphic in our population. The only gain-in-function polymorphism (+489), and -762 promoter polymorphisms failed to reveal differences in genotypic or allelic distributions. The C allele at the +1513 site was identified as a risk factor for TB in this North Indian Punjabi population; the +1729 site was found to be monomorphic, unlike its polymorphic distribution in a South Indian TB patient population.

Association of IL17 and IL23R gene polymorphisms with rheumatic heart disease in South Indian population

ABSTRACTBackground: IL-23/Th17 signaling pathway plays a crucial role in the cell-mediated immune response against bacterial infections and also in the pathogenesis of inflammatory and autoimmune diseases. Recent studies indicate that Th17 cell-associated cytokines are involved in the progression and maintenance of valvular lesions in rheumatic heart disease (RHD). Variants in the genes of cytokines that are potentially involved in Th17 response may influence interindividual differences in their expression levels, thereby contributing to the pathogenesis of immune-mediated diseases such as RHD.Objective: The aim of the study is to investigate the association of IL17A, IL17F, and IL23R gene variants with the risk perception of RHD.Methods: A total of 225 individuals (99 RHD patients and 126 healthy siblings) were recruited for the study. The IL17A (rs2275913), IL17F (rs763780), and IL23R (rs10889677) polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphisms and amplification-refractory mutation system-polymerase chain reaction methods, respectively.Results: The frequency of IL17A (rs2275913) A/A genotype was significantly high in pooled RHD patients (odds ratio [OR] = 2.76; pc = 0.021), rheumatic fever (RF) patients (OR = 14.5; pc = 0.0001), and mitral valvular lesions patients (OR = 2.74; pc = 0.039) when compared to healthy siblings. However, the IL17F (rs763780) and IL23R (rs10889677) polymorphisms did not show any association with RHD.Conclusions: The results suggest that IL17A (rs2275913) polymorphism is associated with the development of RF/RHD in South Indian population. Further studies are required to substantiate the association of these genes with the disease risk.

A Unique Interaction of IVS-I-1 (G>A) (HBA2: c.95+1G>A) with Hb Adana (HBA2: c.179G>A) Presenting as Transfusion-Dependent α-Thalassemia

Nondeletional α-globin mutations are known to cause more serious clinical effects than deletional ones. A rare IVS-I-1 (G>A) (HBA2: c.95+1G>A) donor splice site mutation interferes with normal splicing of pre mRNA and results in activation of a cryptic splice site as well as a frameshift mutation. Hb Adana [HBA2: c.179G>A (or HBA1)] is a highly unstable variant hemoglobin (Hb) resulting from a mutation at codon 59 on the HBA2 or HBA1 gene, recognized to cause severe α-thalassemia (α-thal) syndromes. We report a unique case of compound heterozygosity for these two mutations in a 9-year-old boy who presented with a Hb level of 5.3 g/dL and hepatomegaly at the age of 15 months. He required regular blood transfusions in view of a Hb level of <7.0 g/dL and failure to thrive. He had thalassemic red cell indices and peripheral blood film. The Hb electrophoresis only showed a raised Hb F level (3.3%) and a pre run peak but the Hb H inclusion test was negative. His father had thalassemic red cell indices but a normal Hb level. His mother had almost normal Hb levels and red cell indices. Hb Adana involving the HBA2 gene was detected by mutiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in the proband and his father. DNA sequencing of the HBA2 gene confirmed the IVS-I-1 mutation in the proband and his mother. This case highlighted the unique interaction of the IVS-I-1 mutation with Hb Adana in a young Malay boy presenting with transfusion-dependent α-thal.

Application of Single-Tube Tri-Primer ARMS-PCR to Detect the NFKB1 ATTG Insertion/Deletion Polymorphism.

The -94 ATTG insertion/deletion polymorphism (rs28362491) is an important functional polymorphism in the NFKB1 gene. It has been shown that rs28362491 is associated with many diseases. The purpose of this study was to establish a simple and reliable method to detect the ATTG insertion/deletion polymorphism. On the basis of the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, a single-tube tri-primer ARMS-PCR method was developed to detect the ATTG insertion/deletion polymorphism in 93 samples. The results of the single-tube tri-primer ARMS-PCR method were validated by DNA sequencing. After optimization of the PCR conditions, the single-tube tri-primer ARMS-PCR was established to detect the insertion/deletion polymorphism using agarose gel electrophoresis. In 93 volunteers, the genotype frequencies were 30.1% for Ins/Ins, 19.4% for Del/Del, and 50.5% for Ins/Del, respectively. The results of the single-tube tri-primer ARMS-PCR method were consistent with the results of DNA sequencing. This single-tube tri-primer ARMS-PCR is a reliable, simple, and cost-efficient genotyping method for the detection of the ATTG insertion/deletion polymorphism in the NFKB1 gene.

Rs17042171 at chromosome 4q25 is associated with atrial fibrillation in the Chinese Han population from the central plains

To determine the correlations of single nucleotide polymorphisms (SNPs) with atrial fibrillation (AF) in the Chinese Han population from the central plains. Methods: A total of 168 hospitalized patients, including 56 AF and 112 controls, were recruited in this case-control study. The clinical data were obtained from the medical records. All 5 SNPs, rs337711 in KCNN2, rs11264280 near KCNN3, rs17042171 near PITX2, rs6771157 and rs6795970 in SCN10A, were genotyped using amplification refractory mutation system-polymerase chain reaction or direct sequencing. The χ2 test was used to compare categorical variables and preliminarily examine correlations between the genotype frequencies and AF. Subsequently, a logistic regression model was constructed to determine the associations between the SNPs and AF based on the above screened results. Odds ratios (ORs) and 95% confidence interval (CI) were calculated to assess the strength of the correlations. Moreover, we downloaded the genotype data from the HapMap Project for linkage disequilibrium analysis of rs17042171. Results: AF patients were likely to be of older age and longer left atrial diameter and had more coronary artery disease and higher hypertension compared with the control group (P<0.05). Among the 5 SNPs, the frequency distribution of genotype AA for rs17042171 was significantly different between the AF and control groups (P<0.05). After adjusting for several covariates, there was still a high risk ratio in patients with the AA genotype compared with the AC+CC genotype (OR: 5.591, 95%CI 2.176 to 14.365, P-B<0.008). Similarly, stratification analysis on the AA genotype demonstrated significant differences between rs17042171 and persistent AF. However, there were not significant correlations between AF and the control groups for the other 4 SNPs (P<0.05). Conclusion: Rs17042171, near PITX2 on chromosome 4q25, is associated with AF susceptibility in the Chinese Han population from the central plains, suggesting that this SNP can provide a new strategy for clinical diagnosis in AF patients.

Regulation of Lipocalin-2 oncogene and its impact on gene polymorphisms on breast cancer patients in Jeddah, Saudi Arabia.

Objectives:To identify the impact of Lipocalin-2 (LCN2) gene polymorphisms on breast cancer patients in Jeddah, Saudi Arabia.Methods:It is a case control study in which blood samples of participants from Medical Reference Clinics and King Abdulaziz University Hospital in Jeddah, Saudi Arabia have been taken between 2014 and 2016. This study recruited 128 participants (50% control, 50% patients) and used Tetra-Primer amplification-refractory mutation system-polymerase chain reaction method for the detection of missense SNP (rs11556770). The study measured LCN2 plasma protein expression by enzyme-linked immunosorbent assay technique.Results:The results have shown that 100% of the genotypes were normal allele (G/G). In contrast, the plasma level of LCN2 was considerably elevated among patients as compared to control (p=0.001), and higher in invasive ductal carcinoma patients (p=0.001). The LCN2 protein expression in plasma level was significantly elevated among patients, particularly who demonstrated invasive ductal carcinoma.Conclusion:There is no significant relationship between breast cancer patients and LCN2 gene polymorphisms (rs11556770).

The association of pri-miRNA- 26a1 rs7372209 polymorphism and Preeclampsia susceptibility

ABSTRACTMicroRNAs (miRNAs) are a class of noncoding small RNAs which regulate gene expression through post-transcriptional repression or degradation of messenger RNA. They play very important roles in various biological processes including growth, differentiation, and proliferation, as well as apoptosis, angiogenesis, and metabolism. Therefore, in the present study, we evaluated the possible effect of functional rs7372209C/T polymorphism in the 5ˊ- region of pri-miRNA- 26a1gene on preeclampsia(PE) susceptibility.This case-control study was conducted on 219 PE women and 204 unrelated healthy controls. The amplification refractory mutation system-polymerase chain reaction method was used for rs7372209C/T genotyping.The pri-miRNA- 26a1 rs7372209CT genotype was associated with decreased PE risk (OR, 0.5 [95% CI, 0.3–0.8], P = 0.001). The frequency of rs7372209TT genotype did not differ between two groups. In addition, the pri-miRNA- 26a1 rs7372209 polymorphism was associated with lower risk of PE in dominant model (CT+TT vs CC) (OR, 0.5 [95% CI, 0.4–0.8], P = 0.002). Although there was no significant difference between mild and severe PE women according to rs7372209CT genotype, the differences between mild and severe PE groups with controls remained significant. The frequency of pri-miRNA-26a1 rs7372209CT genotype was not different between late-onset PE and early onset PE groups.The present study showed for the first time that the pri-miRNA- 26a1 rs7372209 polymorphism was associated with lower risk of mild and severe PE in the dominant model and this polymorphism could be a protective factor for PE susceptibility.

Co-existence of BRAFV600E and TERT promoter mutations in papillary thyroid carcinoma is associated with tumor aggressiveness, but not with lymph node metastasis.

BackgroundMutations of BRAFV600E and TERT promoters are associated with thyroid cancer development. This study further investigated association of these mutations with clinicopathological characteristics from patients with papillary thyroid carcinoma (PTC).MethodsTumor tissues from 342 PTC patients were obtained for DNA extraction and polymerase chain reaction amplification to detect the BRAFV600E mutation using amplification-refractory mutation system-polymerase chain reaction. TERT promoter mutations were assessed using Sanger DNA sequencing. The association of these gene mutations with clinicopathological characteristics was then statistically analyzed.ResultsTwo hundred and seventy of 342 (78.9%) PTC patients harbored the BRAFV600E mutation, which was associated with older age male patients. Moreover, TERT promoter mutations occurred in 12 of 342 (3.5 %) PTC patients, all of whom also had the BRAF mutation. One hundred thirty-three patients with papillary thyroid microcarcinoma (PTMC) had no TERT mutations. Statistically, the coexistence of BRAF and TERT promoter mutations were significantly associated with older age, larger tumor size, extrathyroidal extension, and advanced tumor stage, but not with central lymph node metastasis, lateral lymph node metastasis, numbers of lymph node metastasis >5, and numbers of involved/harvested lymph nodes (No. of LNs involved or harvested). The multivariate analyses showed older age (odds ratio [OR], 2.194; 95% CI: 1.117–4.311; p=0.023), larger tumor size (OR, 4.100; 95% CI: 2.257–7.450; p<0.001), and multiplicity (OR, 2.240; 95% CI: 1.309–3.831; p=0.003) were all independent predictors for high prevalence of extrathyroidal extension. However, there was no statistical association with any clinicopathological characteristics except for Hashimoto thyroiditis in PTMC.ConclusionThe current study demonstrated that the coexistence of BRAF and TERT promoter mutations were associated with the PTC aggressiveness, although these mutations were not associated with PTC lymph node metastasis or with PTMC.

The effect of CYP1A2 genotype on the ergogenic properties of caffeine during resistance exercise: a randomized, double-blind, placebo-controlled, crossover study

The purpose of this study was to examine the effect of CYP1A2 -163C>A polymorphism on the ergogenic effects of caffeine supplementation during a resistance exercise (RE) session. In a randomized, double-blind, placebo (PL)-controlled, crossover study, 30 resistance-trained men took part in two RE sessions (three sets to failure at 85% of one repetition maximum, 2-min rest between sets), including bench press (BP), leg press (LP), seated cable row, and shoulder press (SP) following caffeine (CAF) (6mgkg-1) or PL (6mgkg-1 of maltodextrin) ingestion 1h prior to the trial. The number of repetitions was recorded after each set, along with calculation of total number of repetitions for each exercise. Genomic DNA was isolated from the whole blood samples for analyzing the CYP1A2 -163C>A polymorphism through amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Subjects were classified as either AA (n = 14) or AC/CC genotypes (n = 16). The two-way ANOVA with repeated measures revealed differences between AAs and AC/CCs under CAF conditions for repetitions performed in sets 1, 2, and 3 of BP (F(1, 28) = 14.84, P = 0.001, ƞ2 = 0.34), LP (F(1, 28) = 8.92, P = 0.006, ƞ2 = 0.24), SR (F(1, 28) = 17.38, P = 0.0001, ƞ2 = 0.38), and SP (F(1, 28) = 3.76, P = 0.063, ƞ2 = 0.11). CAF also increased the total number of repetitions performed for all three sets in AAs versus AC/CCs for BP (F(1, 28) = 8.72, P = 0.006, ƞ2 = 0.23), LP (F(1, 28) = 4.67, P = 0.03, ƞ2 = 0.14), SR (F(1, 28) = 5.54, P = 0.02, ƞ2 = 0.16), and SP (F(1, 28) = 3.89, P = 0.058, ƞ2 = 0.12) in athletes who were homozygous carriers of the A allele, compared to the C allele carriers. Therefore, AA homozygotes were able to carry out a greater total volume of RE work under CAF but not PL conditions, compared to the C allele carriers. In conclusion, acute ingestion of CAF significantly enhanced RE performance in resistance-trained men who were homozygous for the A allele, but not for C allele carriers. Further studies are needed to replicate the potential role of the CYP1A2 -163C>A polymorphism on the ergogenic effects of CAF in other modes of exercise and in other populations.

Rs4073 single nucleotide polymorphism of interleukin-8 (CXCL8/IL-8) and susceptibility to pulmonary tuberculosis in Gilan, Northern Iran

BackgroundTuberculosis (TB) is an infection in humans that afflicted 10.4 million people in 2015. Interleukin-8 plays an important role in regulating the influx of leukocytes at the site of infection and it is a chemotactic factor for lymphocytes T, neutrophils, and monocytes. ObjectiveIn this study, the relationship between the −251A/T (rs4073) polymorphism of the IL-8 gene and the susceptibility to pulmonary tuberculosis was investigated. MethodsSingle nucleotide polymorphism (SNP) of the IL-8 gene at the position −251A/T was analyzed in 44 patients with pulmonary tuberculosis and 44 healthy controls using the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method. ResultsThere was no significant difference in the frequencies of the alleles and the variant genotypes in the rs4073 SNP of the IL-8 between patients and the control group (P = 0.344 and P = 0.552, respectively). No significant relationship between the frequency of genotype and gender with susceptibility to pulmonary tuberculosis was observed in the study population (Males: P = 0.277 and females: P = 0.615). ConclusionThe SNP (rs4073) in the IL8 gene is not associated with susceptibility to pulmonary TB in the Iranian population.

Association of single nucleotide polymorphisms in miR- 499 and miR-196a with susceptibility to breast cancer

Purpose: To investigate the relationship between miR-499 rs3746444 and miR-196a rs11614913 polymorphisms, and susceptibility to breast cancer in an Iranian population. Methods: This case-control study was performed on a population of 200 subjects comprising 100 breast cancer patients (case/observation group) and 100 healthy individuals (control group). Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used to genotype these polymorphisms. P-values and odd ratios were determined, and p-values 1 were considered statistically significant. Results: There were no significant differences between observation and control groups with respect to rs11614913 T/C polymorphism. The rs11614913 T allele was not identified as a risk factor for susceptibility to breast cancer (OR = 0.86, 95 % CI = 0.85 - 1.3, p = 0.46). However, there were significant differences between observation and control groups with respect to rs3746444 T/C polymorphism. It was observed that cytosine-cytosine (CC) (OR = 4.5, 95 % CI = 1.3 - 15.4, p = 0.06), and cytosine-thymine (CT) (OR = 1.9, 95% CI = 1- 3.6, p = 0.04) genotypes had protective influence against susceptibility to breast cancer. Conclusion: These results indicate that CC and CT genotypes are associated with reduced risk of breast cancer. In particular, the presence of C allele is significantly associated with a low risk of breast cancer. These findings may provide useful information for prevention and early diagnosis of breast cancer. Keywords: Breast cancer, Early diagnosis, miR-196a rs11614913, miR-499 rs3746444, Polymorphism, ARMS-PCR

Molecular Characterization of β-Thalassemia Mutations Via the Amplification Refractory Mutation System-Polymerase Chain Reaction Method at the North Waziristan Agency, Pakistan

β-Thalassemia (β-thal) is a monogenic disease characterized by mutations on the HBB gene, affecting the production of globin that results in hypochromic and microcytic anemia. The aim of this study was to determine the prevalence of six common β-thal mutations, and their frequency and inheritance pattern in affected populations of North Waziristan Agency, Pakistan. In this study, 130 blood samples from 37 unrelated β-thalassemic families having a minimum of one transfusion-dependent child with β-thal major (β-TM), were retrieved either from the Thalassaemia Centre for Women and Children Hospital Bannu or their home towns situated in Noth Waziristan Agency. All samples were analyzed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using six allele-specific primers for the presence of the six β-thal mutations common in the Pakistani population. Of the six common mutations, our study demonstrated five HBB mutations comprising HBB: c.27_28insG, HBB: c.92+5G>C, HBB: c.126_129delCTTT, HBB: c.92+1G>T and HBB: c.17_18delCT from the families studied, while mutation HBB: c.47G>A [codon 15 (G>A)] was not detected in any of the studied families. Furthermore, the HBB: c.27_28insG and HBB: c.92+5G>C were noted to be the most common with frequencies of 42.85 and 31.42%, respectively. The findings of the present study may be useful in launching carrier screening and prenatal diagnosis (PND) programs by screening analyzed and other unanalyzed affected families for the possible presence of common mutations through the ARMS-PCR technique that will help to control the disease.

Preemptive NUDT15 genotyping: redefining the management of patients with thiopurine-induced toxicity.

Thiopurine methyltransferase (TPMT) gene variants have achieved limited success in predicting the outcome of thiopurine therapy, which shows wide inter-individual variations. The literature indicates a strong association between the NUDT15 gene variant and thiopurine-induced toxicity in Asian patients. The present study intends to explore the role of the NUDT15 variant (C415T) in Indian patients on thiopurine therapy. NUDT15 and TPMT genotyping were performed using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) and the restriction fragment length polymorphism (RFLP) technique. Of 370 samples received for TPMT testing, 206 samples were available for NUDT15 genotyping. The NUDT15 risk allele frequency was 10.7%, with the frequency of wild, heterozygous and mutant genotypes being 80.6%, 17.5% and 1.9%, respectively. TPMT variants were seen in 13 of 370 (3.5%) patients, whereas the NUDT15 variant was seen in 40 of 206 (19.4%) patients. Thiopurine-induced toxicity information was available for 101 patients, among whom 10 developed leukopenia and all harbored the NUDT15 variant (p<0.0001). NUDT15 was clinically more relevant than TPMT in terms of sensitivity and specificity, as well as with a statistically significant difference in thiopurine dose requirement for patients with the NUDT15 variant. A preemptive NUDT15 genotyping approach can therefore help identify high-risk patients (NUDT15 C415T positive) who could benefit from thiopurine dose reduction, thereby preventing fatal thiopurine-induced toxicity.

Granulocyte colony-stimulating factor gene rs1042658 variant and susceptibility to idiopathic recurrent pregnancy loss: A case-control study

Granulocyte colony-in stimulating factor (G-CSF) gene can be a potential candidate gene implicated recurrent pregnancy loss (RPL), a common complication of pregnancy with the prevalence of 1-5% among women of reproductive age. To investigate the association between rs1042658 polymorphism in the 3' untranslated region (3'UTR) of G-CSF gene and the risk of unexplained RPL among Iranian women. In total, 122 women with unexplained RPL and 140 healthy postmenopausal women as a control group were enrolled in this case-control study. Tetra-primer amplification refractory mutation system-polymerase chain reaction was performed to determine the rs1042658 genotypes in all subjects. Statistically significant differences were detected between the distribution frequencies of both heterozygote CT, and carriage of T allele (TT+CT) genotypes of the rs1042658 between case and control groups. Allelic association was not observed with RPL. Regarding the results of the present study, G-CSF rs1042658 gene polymorphism could be considered as a probable risk factor for unexplained RPL among Iranian women.

Association of elevated homocysteine levels and Methylenetetrahydrofolate reductase (MTHFR) 1298 A > C polymorphism with Vitiligo susceptibility in Gujarat

BackgroundSeveral studies have reported hyperhomocysteinemia in vitiligo patients, suggesting the potential role of elevated homocysteine levels in precipitating vitiligo. ObjectivesWe aimed to estimate homocysteine and vitamin B12 levels, and to investigate the role of MTHFR 677 C > T and 1298 A > C polymorphisms in vitiligo susceptibility in Gujarat population. MethodsHomocysteine and vitamin B12 levels were estimated in plasma of 55 vitiligo patients and 60 controls by Electrochemiluminescence immunoassay (ECLIA). Polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) techniques were used to genotype MTHFR 677 C > T and 1298 A > C polymorphisms in 520 vitiligo patients and 558 controls. ResultsOur results showed significantly elevated homocysteine levels (p = 0.0003) as well as significant decrease in vitamin B12 levels (p = 0.0102) in vitiligo patients, as compared to controls. No significant difference in genotype and allele frequencies of MTHFR 677 C > T polymorphism was observed among patients and controls, however, the frequency of ‘CC’ genotype of MTHFR 1298 A > Cpolymorphism was significantly increased in patients as compared to controls (p = 0.0151). Analysis based on the type of vitiligo revealed a significant increase in ‘C’ allele of MTHFR 1298 A > C polymorphism in patients with generalized (p = 0.003) and active (p = 0.007) vitiligo as compared to controls. Both the polymorphisms of MTHFR were in low linkage disequilibrium (LD) and susceptible ‘TC’ haplotype was more frequently observed (p = 0.008) in vitiligo patients. Interestingly, elevated homocysteine levels were also positively correlated with MTHFR 1298 A > C polymorphism in vitiligo patients. Structure based in silico prediction revealed structural perturbations in MTHFR protein due to Ala222Val and Glu429Ala amino acid substitution. ConclusionsThe present findings suggest that MTHFR 1298 A > C polymorphism and, altered homocysteine and vitamin B12 levels might play a vital role in the precipitation of vitiligo.

Analysis of Common β-Thalassemia Mutations in North Vietnam

Available and flexible choice of methods for screening and detecting β-thalassemia (β-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common β-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (–TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

Association of genetic polymorphism of interleukin-17A & interleukin-17F with susceptibility of psoriasis.

Background & objectives:Psoriasis is a chronic inflammatory skin disease with unknown aetiology. So far studies have confirmed that interleukins, pro-inflammatory factors and T-cell activation play major role in the development of disease. Interleukin-17 (IL-17) a T helper inflammatory cytokine, was found to be positively correlated with severity of psoriasis. However, the specific mechanism has not been clarified. IL-17A and IL-17F are group members of IL17 family cytokines and found to be located adjacent to one another on the same human chromosome, 6p12. The present study was designed to identify the association between IL-17A and IL-17F gene polymorphism with susceptibility of psoriasis in north Indian population.Methods:A total of 166 psoriasis patients and 150 healthy controls were genotyped for IL-17A and IL-17F gene polymorphism by amplification refractory mutation system-polymerase chain reaction method. One single nucleotide polymorphism (SNP) was analysed in IL-17A (rs10484879) and one SNP in IL-17F (rs763780) to look for an association with psoriasis.Results:Our study indicated decreased frequency of IL-17A (rs10484879) G allele (51.8 vs. 65.0%), and IL-17F (rs763780) C allele (36.5 vs. 45.7%) in psoriatic patients as compared to healthy controls.Interpretation & conclusions:The present findings suggest that IL-17A (rs10484879) G/T and IL-17F (rs763780) C/T gene polymorphisms may contribute in pathogenesis of psoriasis. Further studies need to be done to confirm these findings.