Refractory Mutation System-polymerase Chain Reaction Research Articles

Analysis of ENAM gene single-nucleotide polymorphism rs3796704 with caries susceptibility in young adult Tamil population.

Background:Dental caries is as primeval as humanity, but still, investigations are undergoing regarding the etiopathogenesis behind this multifactorial disease. Genetics is known to play a vital role in the etiology behind dental caries in addition to environmental and socioeconomic factors. Genetic variations like single-nucleotide polymorphisms (SNPs) were extensively studied in the past decade to portray the etiopathogenesis contributing to dental caries.Aim:This investigation was undertaken to analyze the ENAM gene SNP rs3796704 with caries susceptibility in ethnic young adult Tamil population of India.Materials and Methods:Out of 370 patients included, 215 patients belonged to the high caries group (Decayed, Missing and Filled Tooth [DMFT] ≥2) and 155 patients belonged to the low caries group (DMFT ≤ 1). DNA was extracted from the blood of all the individuals. SNP genotyping was performed utilizing tetra-primer amplification refractory mutation system–polymerase chain reaction with specific primers.Results:The genotyping results showed that there were no differences in allelic (P = 0.114) and genotypic frequencies (P = 0.159) between the high caries and low caries groups.Conclusion:Future studies can be conducted in larger samples and different ethnicities around the globe to analyze the role played by SNPs of enamel formation genes in cariogenesis.

Deciphering the Variants Located in the MIR196A2, MIR146A, and MIR423 with Type-2 Diabetes Mellitus in Pakistani Population.

MicroRNAs (miRNAs) are small non-coding RNA molecules that control the post-transcriptional gene expression. They play a pivotal role in the regulation of important physiological processes. Variations in miRNA genes coding for mature miRNA sequences have been implicated in several diseases. However, the association of variants in miRNAs genes with Type 2 Diabetes Mellitus (T2DM) in the Pakistani population is rarely reported. Therefore, the current study was designed to investigate the association of rs11614913 T/C (MIR196A2), rs2910164 G/C (MIR146A), and rs6505162 C/A (MIR423) in clinicopathological proven T2DM patients and gender-matched healthy controls. The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction method was used to determine the genotypes and to establish the association of each variant with T2DM through inherited models. In conclusion, the present study showed that variants rs11614913 T/C and rs2910164 G/C were linked with the risk of T2DM. The data suggested that rs11614913 T/C and rs2910164 G/C could be considered as novel risk factors in the pathogenesis of T2DM in the Pakistani population.

Association between Microalbuminuria and MYH9 Gene Polymorphisms in Hypertensive Iraqi Patients with Metabolic Syndrome

Objective: The presented work aimed to study the correlation between micro-albuminuria and MYH9 (T˃C) (rs3752462) gene polymorphisms in metabolic syndrome of Iraqi patients.
 Methods: This study was a cross - sectional study. Sample size was 140 persons of both gender selected randomly between 1st , April. , 2019 to 15th, Aug. , 2020. They are divided into two groups, 80 with metabolic syndrome patients and 60 with control groups subdivided into another two groups: 30 persons with obese and 30 persons without obese. The distribution of sample according to the gender, the number of males had metabolic syndrome were (47) and female (33), but in obese healthy control the number of males were (19) and female (11), while in non-obese healthy control the number of males were (15) and female (15). The age groups were ranged between (40-60) years. Typing of MYH9 was performed by refractory mutation system-polymerase chain reaction (ARMS-PCR). The current study was performed to investigate the SNPs that affected MYH9 gene which was (T˃C) (rs3752462).
 Results: There was a significant difference between blood sugar, HbA1c, total cholesterol, TG, VLDL-C, LDL-C, blood urea, micro-albuminuria, BMI and HOMA-IR in metabolic syndrome patients as compared with healthy control groups (P ≤ 0.01). The significant result (P ≤ 0.01) were appeared between (TT) for MYH9 genotype patients and obese control with biochemical parameters (Blood glucose, HbA1c, TC, TG, VLDL-C and LDL-C). The correlation between (TT) SNP for MYH9 genotype patients and non - obese control with biochemical parameters is significant result (P ≤ 0.01 and P ≤ 0.05) (Blood glucose, HbA1c, insulin, TC, TG, VLDL-C, LDL-C, urea and micro-albuminuria). The significant result (P ≤ 0.01 and P ≤ 0.05) were appeared between (CT) for MYH9 genotype patients and obese control with biochemical parameters (Blood glucose, HbA1c, TC, TG and VLDL-C) respectively.
 Conclusion: There is a significant difference between blood glucose, HbA1c, total cholesterol, TG, VLDL-C, LDL-C, blood urea, micro-albuminuria, BMI and HOMA-IR in metabolic syndrome patients and healthy control groups (P ≤ 0.01). Also a significant result (P ≤ 0.01) obtained between (TT) for MYH9 genotype patients and obese and non-obese control with some biochemical parameters. (P ≤ 0.01 and P ≤ 0.05). The significant result (P ≤ 0.01 and P ≤ 0.05) appear between (CT) for MYH9 genotype patients and obese control with some biochemical parameters.

Protective role of TIRAP functional variant against development of coronary artery disease.

Coronary artery disease (CAD) is the leading cause of sudden death worldwide. Inflammation is proved to be an important player in development of the CAD. Inflammation is directly regulated by the Toll like receptors (TLRs). Susceptibility of CAD is influenced by genetic variations within TLRs and the proteins involved in its signaling cascade. The TIRAP/MAL {TIR domain containing adaptor protein / MyD88 (myeloid differentiation primary response gene 88) adaptor-like} exhibits maximum genetic variations of all adaptor proteins involved in TLR signaling cascade. Susceptibility to a number of diseases can be influenced due to presence of S180L single nucleotide polymorphism (SNP) of TIRAP/MAL. This study was conducted to investigate the functional role of this well characterized S180L polymorphism on susceptibility to CAD among Pakistani patients. A total of 146 Pakistani CAD patients and 147 controls were genotyped by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) and the data was analyzed by using 2-tailed Chi square (x2) test. The p value ≤ 0.05 was considered to be significant.Significantly high frequency of homozygous L180L genotype was observed among healthy subjects as compared to the CAD patients [24 (16%) vs 7 (5%); x2 11.85; p = 0.003]. Moreover, the allele frequency of the minor allele; 180L was observed to be significantly higher among controls than the CAD patients, having same direction of association [156 (53%) vs 131 (45%); OR (95% CI) = 0.7198 (0.520–0.996); p < 0.05).Our results indicate that protective effect of L180L; a coding variant of TIRAP/MAL against CAD is discernible.

Correlations and Influence of TAP2 Genes Polymorphisms and Systemic Lupus Erythematosus Propensity.

The present study was designed to evaluate the association of transporters associated with antigen processing (TAP2) polymorphisms TAP2-379Ile > Val (rs1800454), TAP2-665Thr > Ala (rs241447) and TAP2-565Ala > Thr (rs2228396) as a candidate gene with susceptibility to the Systemic Lupus Erythematosus (SLE). To analyze these three polymorphic variants, 88 patients with SLE and 100 healthy controls from northeastern Iran were enrolled from May 2018 to July 2019. Genomic DNA polymorphisms were performed by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. Data were analyzed by SPSS software. In this cross-sectional study, there was a stratification between patients and controls. The distribution of the frequency of Ala73 (41.5%) allele at TAP2/665 and Ile 19 (10.8%) allele at TAP2/379 was higher in patients. Additionally, the Ala/Ala 13(14.8%) and Ala/Thr 49(55.7%) genotypes distributions at 665 positions were higher in SLE patients compared to the controls. Furthermore, frequencies of TAP2*H allele significantly increased in SLE patients 10(5.71%) (P=0.01). Frequency of TAP2*A allele in the control group was 120(60%) (p=0.06) due to the dominant genetic model. This allele has a protective effect against SLE. There was no relationship between TAP2*D, TAP2*E, TAP2*F and TAP2*G alleles with the outbreak of SLE. Our data indicated that genetic variants in TAP2 gene may be associated with SLE disease. A correlation between Ala allele at TAP2/665 and Ile allele at TAP2/379 polymorphisms and pathogenesis of SLE was observed.

Evaluation of the GJB2 and GJB6 Polymorphisms with Autosomal Recessive Nonsyndromic Hearing Loss in Iranian Population.

Introduction:Hearing loss (HL), with more than 100 gene loci, is the most common sensorineural defects in humans. The mutations in two GJB2 and GJB6 (Gap Junction Protein Beta 2, 6) genes are responsible for nearly 50% of autosomal recessive nonsyndromic hearing loss. The aim of the present study was to evaluate polymorphisms of 111C>T (rs7329857) and 337G>T (rs7333214) in GJB2 (encoding connexin 26) and GJB6 (encoding connexin 32) genes, respectively.Materials and Methods:In this study, 32 blood samples were obtained from Iranian patients with HL defect and 32 normal blood samples were prepared. After genomic deoxyribonucleic acid extraction, genotyping in rs7333214 and rs7329857 polymorphisms was conducted using tetra-amplification refractory mutation system-polymerase chain reaction and the obtained data were analyzed.Results:In this study, the prevalence rates of CC, CT, and TT genotypes in GJB2 gene were reported as 84.4%, 68.7%, and 0% in the affected subjects and 0%, 15.6%, and 31.3% in the control samples, respectively, which were statistically significant (P=0.004). In relation to GJB6 gene, the prevalence rates of GG, GT, and TT genotypes were 65.2%, 78.1%, and 25% in the control subjects and 21.9%, 9.4%, and 0% in the affected samples, respectively, which were not statistically significant (P>0.05).Conclusion:The results of this study revealed that 111C>T polymorphism in GJB2 gene was involved in the incidence of HL in the studied population and could be suggested as a prognostic factor in genetic counseling before marriage and pregnancy.

Frequencies of Beta Thalassemia Mutations Show Different Pattern in Bannu Region than Other Parts of Pashtun Population in Khyber Pakhtunkhwa Province Pakistan.

β-Thalassemia arises as result of mutations in HBB gene, influencing the globin production which results in hypochromic and microcytic anaemia. The present study was aimed to investigate the occurrence of six common β-thal mutations, its inheritance pattern, frequency, and consanguinity in parents of Bannu region Khyber Pakhtunkhwa (KP) province, Pakistan. Conducting such studies may impart important information about thalassemia prevention like prenatal diagnosis (PND), carrier screening and genetic counselling which may be helpful in controlling the suspected births. During the study, 250 blood samples were retrieved from different families comprising of one transfusion dependent child and sporadic patients from different areas of Bannu region. The collected blood samples were investigated to see if there is any common mutations which may trigger β-Thalassemia employing amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) approach. Amongst the studied mutation in District Bannu, frame shift codons (FSC) 8/9 (+ G) (HBB: c.27_28insG) was observed to be the most common mutation followed by Codons 41/42 (- TTCT), IVS-I-5(G > C) and FSC 5 (- CT) having frequencies of 42, 26, 19 and 13 respectively. The results obtained by the present study were found different from previous studies demonstrated from other Pashtun regions of KP, showing heterogeneity in frequencies of known mutations. These observations may help in implementing parental meetings about disease recurrence in future, large scale mutation screening, and prenatal diagnosis in the whole Pashtun ethnicity including District Bannu.

Development of a mutation hotspot detection kit for the phenylalanine hydroxylase gene by ARMS-PCR combined with fluorescent probe technology.

To develop a screening kit for detecting mutation hotspots of the phenylalanine hydroxylase (PAH) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with phenylketonuria (PKU) diagnosed during neonatal genetic and metabolic disease screening in Shaanxi province, and their mutations were analyzed. We designed and developed a screening kit to detect nine mutation sites covering more than 50% of the PAH mutations found in Shaanxi province (c.728G>A, c.1197A>T, c.331C>T, c.1068C>A, c.611A>G, c.1238G>C, c.721C>T, c.442-1G>A, and c.158G>A) by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) combined with fluorescent probe technology. Peripheral blood and dried blood samples from PKU families were used for clinical verification of the newly developed kit. PAH gene mutations were detected in 84 children diagnosed with PKU. A total of 159 mutant alleles were identified, consisting of 100 missense mutations, 28 shear mutations, 24 nonsense mutations, and 7 deletion mutations. Exon 7 had the highest mutation frequency (32.08%). Among them, the mutation frequency of p.R243Q was the highest, accounting for 20.13% of all mutations, followed by p.R111X, IVS4-1G>A, EX6-96A>G, and p.R413P; these five loci accounted for 47.17% (75/159) of all mutations. In addition, we identified three previously unreported PAH gene mutations (p.C334X, p.G46D, and p.G256D). Fifteen mutation sites were identified in the 47 PAH carriers identified by next-generation sequencing (NGS), which were verified by the newly developed kit, with an agreement rate of 100%. This newly developed kit based on ARMS-PCR combined with fluorescent probe technology can be used to detect common PAH gene mutations.

Association of P2X7 receptor genetic polymorphisms and expression with rheumatoid arthritis susceptibility in a sample of the Iranian population: a case-control study.

Rheumatoid arthritis (RA) is a complex inflammatory autoimmune disease with joint eruption, systemic manifestation, and numerous predisposing genetic factors. The P2X7 receptor is an essential ligand-gated channel that contributes to many physiological processes, especially inflammation. However, genetic variations can alter the P2X7 receptor function. Therefore, the present study aimed to explore the impact of P2X7 genetic polymorphisms and expression on susceptibility to RA in a sample of the Iranian population. We enrolled 160 (145 female, 15 male) RA patients and 160 (142 female, 18 male) healthy individuals in this study. Genotyping was performed using tetra amplification refractory mutation system-polymerase chain reaction (TARMS-PCR) for rs1718119, rs2230912, rs2393799, rs28360457, rs35933842, and allele-specific PCR for rs1653624 and rs3751143. Furthermore, 44 new cases of RA and 48 healthy controls were recruited to investigate whether P2X7 mRNA expression is associated with RA susceptibility. The results revealed that the rs2393799 significantly increased the risk of RA in all genetic models (p<0.05), while rs3751143 in codominant (CC vs. AA, OR=0.49, 95% CI=0.26-0.92), dominant (AC+CC, OR=0.59, 95% CI=0.37-0.94), C allele (OR=0.63, 95% CI=0.46-0.88), and rs2230912 in codominant (AG vs. AA, OR=0.56, 95% CI=0.34-0.94), dominant (AG+GG vs. AA, OR=0.59, 95% CI=0.35-0.99), and overdominant (AG vs. AA+GG, OR=0.57, 95% CI=0.33-0.98) significantly decreased the RA risk (p<0.05). Furthermore, the rs1718119 and rs1653624 were not associated with susceptibility of RA (p>0.05), and rs28360457 and rs35933842 were not polymorphic in our study. The mRNA expression level of P2X7 in both groups revealed that the P2X7 gene was significantly upregulated in RA (3.18±0.43) compared to healthy subjects (1.47±0.15, p<0.001). Our results suggest that rs2393799, rs3751143, and rs2230912 variants of the P2X7 gene are associated with RA's susceptibility in a sample of the Iranian population. Also, P2X7 mRNA expression was higher in our new RA patients. The P2X7 receptor has been considered as a potential pharmacologic target in RA. Key Points • P2X7 variants (rs2393799, rs2230912, rs3751143) were associated with RA susceptibility in a sample of the Iranian population. • rs2393799 increases the risk of RA, while rs2230912 and rs3751143 decrease the risk of RA. • P2X7 expression was significantly upregulated in new RA patients compared to controls.

Investigating the CYP2B6 rs3745274 and rs3211371 polymorphisms in Methadone-Responder and Non-Responder Addicts in Iran.

Methadone therapy is a major protocol in opioid addiction cases in many health care systems. Population-based studies have shown that in addicted people, the genetic profile affects their response to methadone therapy. Therefore, this study designed to examine the frequency of two SNPs of the CYP2B6 gene (rs3745274 and rs3211371) in addicted cases in two methadone-responders and methadone non-responders groups. A total of 199 opioid-addicted individuals and 117 unaffected control subjects were genotyped for rs3745274 and rs3211371 polymorphisms of the CYP2B6 gene using the tetra-primer amplification refractory mutation system-polymerase chain reaction. Results of this study revealed the significant association of rs3745274 GG (p < 0.001; OR = 0.027; 95% CI = 0.14-0.49) and GT (p < 0.001; OR = 4.04; 95% CI = 2.26-7.21) genotypes with the risk of addiction in methadone-responders. Also, a significant association between rs3745274 GG (p < 0.001; OR = 0.28; 95% CI = 0.15-0.51) and GT (p < 0.001; OR = 5.1; 95% CI = 2.8-5.28) genotypes and addiction relapse was found in methadone non-responders. Based on our findings, we can conclude that rs3745274 variant of CYP2B6 gene could serve as a potential biomarker, to evaluate the prognosis of addicted people fate under treatment with methadone.

Long non-coding RNA H19 expression and functional polymorphism rs217727 are linked to increased ischemic stroke risk

BackgroundEfforts to identify potential biomarkers for the diagnosis of ischemic stroke (IS) are valuable. The H19 gene plays a functional role in increasing the prevalence of IS risk factors. We evaluated the correlation between H19 rs217727 polymorphism and the expression level of H19 lncRNA with susceptibility to IS among the Iranian population.MethodsBlood samples were collected from IS patients (n = 114) and controls (n = 114). We concentrated on the expression pattern of H19 at different time points (i.e., 0–24, 24–48, and 48–72 h after stroke). The tetra-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was applied for DNA genotyping. We used the quantitative real-time PCR to evaluate H19 expression levels. We used the receiver operating characteristic (ROC) curve to evaluate the diagnosis and prognosis of IS.ResultsThe rs217727polymorphism of H19 was related with IS susceptibility in the co-dominant (OR = 2.92, 95% CI = 0.91–10.92, P = 0.04) and recessive models (OR = 2.80, 95% CI = 0.96–8.15, P = 0.04). H19 expression was significantly upregulated in IS and remained high for 72 h after stroke. ROC curves showed that H19 expression within the first 24 h from stroke onset might serve as a biomarker for the early diagnosis of IS with 79.49% sensitivity and 80.00% specificity. H19 expression in small vessel occlusion (SVO) and large-artery atherosclerosis (LAA) patients were 3.74 and 3.34 times higher than the undetermined (UD) subtype, respectively [OR = 3.74 95% CL (1.14–12.27) P = 0.030 and OR = 3.34 95% CL (1.13–9.85) P = 0.029].ConclusionThe rs217727 polymorphism of the H19 is correlated with IS susceptibility, and H19 expression levels were higher in SVO and LAA patients. The upregulation of H19 may be considered as a diagnostic biomarker in IS among the Iranian population, but it cannot serve as a useful prognostic marker.

Prevalence and distribution of major β-thalassemia mutations and HbE/β-thalassemia variant in Nepalese ethnic groups

BackgroundBeta-thalassemia is a genetic disorder that is inherited in an autosomal recessive pattern. This genetic disease leads to a defective beta-globin hemoglobin chain causing partial or complete beta-globin chain synthesis loss. Beta-thalassemia major patients need a continuous blood transfusion and iron chelation to maintain the normal homeostasis of red blood cells (RBCs) and other systems in the body. Patients also require treatment procedures that are costly and tedious, resulting in a serious health burden for developing nations such as Nepal. MethodsA total of 61 individuals clinically diagnosed to have thalassemia were genotyped with multiplex amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Twenty-one major mutations were investigated using allele-specific primers grouped into six different panels. ResultsThe most common mutations found (23%) were IVS 1–5 (G-C) and Cd 26 (G-A) (HbE), followed by 619 deletion, Cd 8/9 (+G), Cd 16 (–C), Cd 41/42 (–TTCT), IVS 1–1 (G–T), Cd 19 (A-G), and Cd 17 (A-T) at 20%, 12%, 8%, 6%, 4%, 3%, and 1%, respectively. ConclusionThe results of this study revealed that Nepal's mutational profile is comparable to that of its neighboring countries, such as India and Myanmar. This study also showed that thalassemia could be detected across 17 Nepal's ethnic groups, especially those whose ancestors originated from India and Central Asia.

Evaluation of the High Resolution Melting Approach for Detection of β-Thalassemia Gene Mutations

β-Thalassemia (β-thal), an autosomal recessive hemoglobinopathy, is one of the most common genetic disorders in Pakistan. Awareness of this disease, genetic counseling, extended family carrier screening and prenatal diagnosis (PND) are helpful in prevention and control. Currently, direct DNA sequencing and multiple amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) are the methods used to detect β-thal mutations, the latter being the most widely used. This study aimed to evaluate PCR-high resolution melting (PCR-HRM) analysis for the detection of most common β-thal mutations that are found in Pakistan. This study was designed to identify the β-thal mutations using PCR-HRM analysis in a total of 90 samples [blood and chorionic villus sampling (CVS)]. These samples were first screened for routine mutations by MARMS-PCR and then evaluated by PCR-HRM analysis. The results of PCR-HRM analyses were further confirmed by direct DNA sequencing and all analyses interpreted the same results in all 90 samples. Eleven cases (36.6%) were detected to carry IVS-I-5 (G>C) (HBB: c0.92 + 5G>C), six cases (20.0%) with frameshift codons (FSC) 41/42 (–TTCT) (HBB: c.126_129delCTTT), five cases (16.0%) were diagnosed with codon 15 (G>A) (HBB: c.47G>A), three cases (10.0%) were found with codon 30 (G>C) (HBB: c.93G>C), one case was diagnosed with FSC 16 (–C) (HBB: c.51delC), one with IVS-I-1 (G>T) (HBB: c0.92 + 1G>T) and one with codon 5 (–CT) (HBB: c.17_18delCT). The PCR-HRM analysis represents a less tedious and more useful method for the detection of β-globin gene mutations.

Role of platelet endothelial aggregation receptor 1 polymorphisms in idiopathic thrombocytopenic purpura: Is there an association?

Background: Genetic risk factors are implicated in the etiology and pathogenesis of immune thrombocytopenic purpura (ITP). Platelet endothelial aggregation receptor 1 (PEAR1) plays an important role in regulating megakaryopoiesis and thrombopoiesis. rs12041331 and rs12566888 single-nucleotide polymorphisms of PEAR1 are associated with megakaryocyte differentiation and platelet function. Materials and Methods: To conduct this study, 68 peripheral blood samples of patients with ITP (56 acute and 12 chronic) were collected. The amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used to detection of rs12041331 and rs12566888 PEAR1 polymorphisms. Results: Statistically significant differences were not seen between rs12041331 and rs12566888 genotypes in acute and chronic groups (P = 0.778, P = 0.844). The frequency of rs12041331 AG/AA genotypes and the rs12566888 GT genotype was more in acute ITP patients; on the other hand, the rs12566888 TT genotype was more in the chronic group. The highest platelet counts and platelet distribution width (PDW) were related to the rs12041331 AG allele. GT and TT of rs12566888 had more PDW and platelet count, respectively. Mean platelet volume values between alleles of both the polymorphisms were constant and did not differ much. In general, no statistically significant differences were observed between genotypes of polymorphisms and platelet parameters. Conclusions: There was no association between rs12041331 and rs12566888 with platelet parameters in ITP patients and the severity of this disease. Further investigation with a larger size is recommended.

A Tetra-Primer Amplification Refractory Mutation System–Polymerase Chain Reaction (T-ARMS-PCR) for Genotyping of rs8099917 &amp; rs12979860 IL28B Polymorphisms and Its Correlation of Various Variables in Iranian HCV Patients

Background: Selecting patients for new direct acting antiviral treatment of HCV has prompted a conflicting matter worldwide because of its high cost and limited availability. Genotyping of IL28B polymorphisms will aid clinical decision making for identifying priorities of urgent treatment in resource-limited countries.&#x0D; Objectives: The aim of the present study was to design a simple tetra-primer amplification refractory mutation system–polymerase chain reaction (T-ARMS-PCR) for genotyping of the rs8099917 and rs12979860 IL28B gene polymorphisms. Furthermore, we identify the correlation of variables such as gender, serum ALT level, histology of liver and baseline viral load with these polymorphisms.&#x0D; Patients and Methods: We efficiently designed a T-ARMS-PCR for detection of rs12979860 and rs8099917 IL28B gene polymorphisms. Using this method, we genotyped 148 hepatitis C patients. To ensure T-ARMS genotyping quality, we, regenotyped samples with the PCR- sequencing method.&#x0D; Results: Results of genotyping of rs12979860 and rs8099917 by T-ARMS PCR method were 100% concordant with the sequencing results. Among these 148 patients with chronic hepatitis C, the frequency of the rs12979860 CT, TT and CC genotypes was 72.3%, 14.2% and 13.5%, respectively and the frequency of the rs8099917 TT, GT and GG genotypes was 58.1%, 38.5% and 3.4%. Low frequency (2.7%) of association of two unfavourable homozygous genotypes (TT rs12979860 / GG rs809917) as well as 56.7% of association of 3 or 4 favorable alleles could explain good response of Iranians to HCV treatment with interferon-based regimens. About correlation of polymorphisms with different variables, only high viral load showed a statistically significant correlation to unfavorable genotype of TT rs12979860 ( p value = 0/05 ) and there was no correlation of serum ALT level, gender and histology of liver to IL28B genotypes.&#x0D; Conclusions: We report that rs8099917 polymorphisms could predict outcomes better than rs12979860 in Iranian HCV patients.

Detection of association of IL1β, IL4R, and IL6 gene polymorphisms with cervical cancer in the Bangladeshi women by tetra-primer ARMS-PCR method

BackgroundCervical cancer (CC) is the main cause of cancer-related deaths among women in developing countries. It is the second leading female malignancy in Bangladesh in terms of incidence and mortality. Our present study aimed to investigate the association of IL1β (rs16944), IL4R (rs1801275), and IL6 (rs1800797) gene polymorphisms with the susceptibility of cervical cancer. Materials and MethodsThis case-control study was conducted on 252 cervical cancer patients and 228 healthy volunteers, using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). ResultsIn the case of rs16944 polymorphism, GG genotype (OR = 2.10, 95%CI = 1.24–3.56), dominant model (OR = 1.71, 95% CI = 1.11–2.63), recessive model (OR = 1.54, 95% CI = 1.01–2.35), and G allele (OR = 1.30, 95% CI = 1.005–1.68) were significantly associated with increased cervical cancer risk. Among these, GG genotype and dominant model remained significant after the Bonferroni correction (p < 0.017). For rs1801275 polymorphism, GG genotype (OR = 2.66, 95% CI = 1.49–4.75), dominant model (OR = 1.49, 95% CI = 1.04–2.14), recessive model (OR = 2.45, 95% CI = 1.40–4.27), and G allele (OR = 1.59, 95% CI = 1.21–2.10) significantly elevated the risk of cervical cancer but significance did not exist for dominant model after the Bonferroni correction. rs1800797 variant showed significantly increased risk in all genetic models including, AG genotype (OR = 8.13, 95% CI = 5.27–12.55), AA genotype (OR = 9.86, 95% CI = 2.76–35.21), dominant model (OR = 8.25, 95% CI = 5.40–12.60), recessive model (OR = 4.41, 95% CI = 1.25–15.56), and A allele (OR = 4.99, 95% CI = 3.49–7.13) and the significances were consistent with the Bonferroni correction except recessive model. Haplotyping analysis indicates that GAA (p = 5.15x10−5) and GAG haplotypes (p = 4.72x10−9) significantly decreased the risk of CC, whereas AAA (p = 3.89x10-9), AAG (p = 0.0003), AGA (p = 3.98x10-5) and AGG haplotypes (p = 0.002) significantly increased the risk of CC. The IL1β mRNA level was up-regulated, which was associated with poor prognosis in silico. ConclusionOur results conclude that rs16944 (IL1β), rs1801275 (IL4R), and rs1800797 (IL6) polymorphisms are associated with cervical cancer in Bangladeshi women.

Impact of JAK2V617F Mutational on Haematologic Features in Sudanese Patients with Essential Thrombocythemia and Thrombotic Risk Assessment

Objective: We correlated selected haematological parameters in Sudanese essential thrombocythemia (ET) patients based on their homozygous/heterozygous JAK2V617F genotype, as well as the application of thrombotic risk assessment using different thrombotic risk scoring models. Methods: In this single-center study, we evaluated 60 patients with ET at the time of the diagnosis without any prior treatment. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique was used to determine JAK2V617F mutation status. Complete blood count was evaluated using the Sysmex analyzer. Furthermore, the thrombotic risk assessment of ET patients using different thrombotic risk scoring models was applied. Results: The JAK2V617F mutation was detected in 29/60 patients (48.3%), of whom 23 (38.3% of total) were heterozygous and 6 (10.0%) were homozygous. Compeered to JAK2 wild-type or JAK2 heterozygous patients, JAK2 homozygous patients for JAK2V617F mutation were associated with older age(p < 0.05), significantly higher mean leukocytes count (P =0.001), significantly lower Hb concentration (p < 0.05), and splenomegaly (p < 0.05), while the mean of the platelet counts was slightly higher, although not reached a significant level. We also found two patients who developed thrombotic events throughout follow-up and were initially classified as a low-risk category in the traditional classification. One of them with age < 60 years, hypertension, and JAK2 homozygosity but without thrombosis history, was allocated in a high-risk category by IPSET-t and r- IPSET-t scores. The second patient was stratified in a low-risk category by all scoring models with age < 60 years, hypertension, leukocytosis, unmutated JAK2, and without a history of thrombosis. Conclusions: The JAK2 V617F homozygosity correlated with older age, higher leukocyte count, lower Hb concentration, and a higher risk of thrombosis in Sudanese ET patients. Evaluation of hypertension and identification of JAK2 V617F homozygosity at diagnosis of ET might give the clinician more meaningful prognostic information and so improve the therapeutic management.

Association analysis of -429T/C receptor for advanced glycation end products (RAGE) gene polymorphism with type 2 diabetic retinopathy and serum soluble RAGE levels in Pakistani patients.

To investigate the association of receptor for advanced glycation end products gene polymorphism 429T/C (rs1800625) with diabetic retinopathy and serum soluble receptor for advanced glycation end products levels in patients with type 2 diabetes. The case-control study was conducted from January 2017 to December 2018 at Pakistan Railway Hospital, Rawalpindi, and the Multidisciplinary Laboratories of Islamic International Medical College, Riphah International University (RIU), Islamabad, Pakistan. Those included were healthy controls in group A, diabetics without retinopathy in group B and patients having diabetic retinopathy in group C. Genotyping for 429T/C was done by tetra-primer amplification refractory mutation system-polymerase chain reaction. Serum soluble receptor for advanced glycation end products levels were measured using enzyme-linked immunosorbent assay. Data was analysed using SPSS 22. Of the 450 subjects, 150(33.3%) were in each of the three groups. The frequency of TT, TC and CC genotypes of 429T/C polymorphism were 137(91.3%), 10(6.7%) and 3(2%) in group A; 133(88.6%), 13(8.7%) and 4(2.7%) in group B; and 127(84.7%), 18(12%) and 5(3.3%) in group C. No significant association of 429T/C genotypic and allelic frequencies were found with groups B and C (p>0.05). Serum soluble receptor for advanced glycation end products levels were significantly high in patients with proliferative diabetic retinopathy and were positively correlated with fasting plasma glucose in group C (p<0.05). TC and CC genotypes were significantly associated with raised serum soluble receptor for advanced glycation end products, and TC with raised fasting plasma glucose in group C. The 429T/C receptor for advanced glycation end products gene polymorphism was found to be associated with severe non-proliferative diabetic retinopathy, and serum soluble receptor for advanced glycation end products levels had a positive correlation with severity of diabetic retinopathy.

TGF-β1 Gene Polymorphism and Its Correlation with Serum Level of TGF-β1 in Psoriasis Vulgaris Among Iraqi People.

PurposeMany cytokines have been implicated in the pathogenesis of psoriasis, among these the transforming growth factor-beta 1 (TGF-β1) can be endorsed by different mechanisms besides inhibiting keratinocytes proliferation. The role of genetic polymorphisms of TGF-β1 has been studied in various inflammatory diseases. Our aim is to study the correlation of TGF-β1 gene polymorphism at codon 10 and 25 with the expression of serum level of TGF-β1 in a sample of Iraqi psoriatic patients compared to the control group.Materials and MethodsA cross-sectional study involved 100 patients with psoriasis vulgaris and 50 sex- and age-matched healthy volunteers as control group. Serum and genomic DNA were prepared from peripheral blood samples. Amplification refractory mutation system–polymerase chain reaction technique (ARMS-PCR) had been applied for genotyping TGF-β1 codon 10 [rs1982073] and codon 25 [rs1800471] genetic polymorphisms. Enzyme-linked immunosorbent assay technique (ELISA) based on the sandwich principle was used for quantification of serum TGF-β1 level. Psoriasis Area and Severity Index (PASI) scoring was applied for determining the severity in psoriatic patients and classified accordingly to mild (PASI<7), moderate (PASI 7–12), severe (PASI>12) groups.ResultsStatistically significant difference was found in TGF-β1 gene polymorphism between psoriatic patients and control group at codon 10 (T869C) polymorphism (p=0.021) and codon 25 (G915C) polymorphism (p=0.040). No significant association was detected with the mean serum TGF-β1 level, severity of the disease, disease onset, gender, history of psoriatic arthritis, and smoking in both codons. Significant lower mean serum TGF-β1 level was found among psoriatic group (192.17 ± 531.12 ng/L) compared with controls (565.89 ± 1372.30 ng/L) (p = 0.018). Relation of mean serum TGF-β1 level with the onset of the disease was statistically significant (p = 0.004), early-onset disease group was lower (105.92 ± 68.02 ng/L) compared with the late-onset disease group (450.92 ±1027.79 ng/L). The mean serum TGF-β1 level showed no significant differences with the severity of psoriasis, gender, history of psoriatic arthritis, and smoking.ConclusionIraqi population showed a significant association between TGF-β1 gene polymorphism at codon 10 and 25 were with psoriasis susceptibility, and a significantly lower mean serum TGF-β1 level was detected in psoriatic patients.

Evaluating the Possible Association between PD-1 (Rs11568821, Rs2227981, Rs2227982) and PD-L1 (Rs4143815, Rs2890658) Polymorphisms and Susceptibility to Breast Cancer in a Sample of Southeast Iranian Women.

Introduction:Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a critical role as a regulator of immune-system cells, including T cell, natural killer T (NKT), monocytes, dendritic cells (DC), and B cells. Objective:This study aimed to find a possible association between PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) variants and Breast Cancer (BC) risk in a sample of southeast Iranian women. Method:The case-control study consisted of 520 individuals, including 260 histologically confirmed BC patients and 260 non-cancer age-matching healthy women as the control group. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) methods were used for genotyping of PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) polymorphisms. Results and Conclusion:Our findings indicated that the PD-L1 rs4143815 (G/C) variant meaningfully reduced the risk of BC. However, the PD-L1 rs2890658 variant increased the BC risk in the AC genotype as well as the A allele. Furthermore, we could not find a meaningful association between PD-1 rs11568821, PD-1 rs2227981, PD-1 rs2227982, and BC. Our team examined the possible association between variants and clinicopathological characteristics, including age, size of tumour, lymph node, histology, grade of tumour, estrogen and progesterone receptors status as well as human growth factor receptor 2 (HER2). Our findings demonstrated that PD-L1 rs4143815, PD-L1 rs2890658, PD-1 rs2227982 had a significant association with age. Additionally, we found a significant relation between PD-1 rs2227982 variant and tumour size. Statistical analyzes of PD-1 rs2227981 and PD-1 rs11568821 variants showed a meaningful relation between tumour grade and tumour stage (p=0.006), respectively.