Polymerase Chain Reaction-amplification Refractory Mutation Research Articles

Association of Polymorphisms in IL-10, TGF-β1, IFN-γ, and TNF-α Genes with the Susceptibility to Chronic Obstructive Pulmonary Disease in Kerman, Iran

Background: One of the principal cigarette smokes (CS) mediated diseases is chronic obstructive pulmonary disease (COPD). Methods: In the current case-control study, the relationship between the polymorphisms of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1 ) codon 10, TGF-β1 codon 25, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in 213 individuals with COPD and susceptibility to the disease, with 100 healthy age and gender-matched people as a control group, was investigated using PCR-ARMS (polymerase chain reaction-amplification refractory mutation system). Moreover, the combination of the polymorphisms of TGF-β1 codon 10.25 regarding this susceptibility was studied in the same condition. Results: There was a significant difference between polymorphism of TGF-β1 codon 10 (+869 T/C), codon 25 (G+915C), and susceptibility to the disease (OR=0.50; (95 %CI=0.24-1.07, p=0.05), ORCC =5.31; (95% CI: 1.22-23.2); p=0.02), thus polymorphism of IL-10 and TGF-β1 increased the risk of susceptibility to COPD but the polymorphisms of TNF-α (G-308A) and IFN-γ (+847 T/A) did not show any association. Conclusion: All in all, it is recommended that the patients carrying the above-said genotypes should be paid proper attention, especially those who are exposed to chemicals at their workplaces, pollution, and cigarette smoke.

Association between a genetic variant in scavenger receptor class B type 1 and its role on codon usage bias with increased risk of developing coronary artery disease

ObjectiveCoronary artery disease (CAD) as an important cause of morbidity and mortality globally. The scavenger receptor class B type 1 (SCARB1) plays an essential role in the reverse cholesterol transport. We have explored the association between a genetic variant, rs5888, in the SCARB1 gene with CAD and serum HDL-C levels. MethodsPatients were categorized into two groups' angiogram positive (>50% coronary stenosis) and angiogram negative (<50% coronary stenosis). Genotyping was carried out using polymerase chain reaction-amplification refractory mutation system. The association between the SNP rs5888 and serum HDL-C was analyzed using a logistic regression model. ResultsThe results showed that the subjects carrying a T allele was associated with a decreased serum HDL-C levels compared to the C allele in total population (p < 0.001). The risk of angiogram positivity in subjects carrying a T allele was 3.1-fold higher than for the control group (p < 0.001). ConclusionCVD patients carrying the T allele of rs5888 variant in the SCARB1 gene was associated with decreased serum level of HDL.

Interaction between a genetic variant in vascular endothelial growth factor with dietary intakes in association with the main factors of metabolic syndrome

IntroductionVascular Endothelial Growth Factor (VEGF) is involved in several biological processes, including angiogenesis and VEGF high levels results in higher risk of metabolic syndrome (MetS) and cardiovascular disease (CVD). We aimed to examine the interaction of the rs10738760 (A > G), a genetic polymorphism of VEGF, and dietary intake in subjects with MetS recruited as part of the Mashhad Stroke and Heart Atherosclerotic Disorders (MASHAD) cohort study. MethodsIn this cross-sectional study, International-Diabetes-Federation criteria (IDF) were applied to determine the presence of MetS. The polymerase chain reaction-amplification refractory mutation system (ARMS-PCR) was used for genotyping. A food frequency questionnaire (FFQ) was used for determining dietary macro/micronutrient intake. ResultsSubjects with the AA genotype for the VEGF polymorphism, and a high consumption of sugar, starch, monounsaturated fatty acids (MUFA), and saturated fatty acid (SFA) showed a significantly higher risk of MetS. There was a significant association between AA genotype with sugar (OR:1.02, 95% CI:1.01–1.03) and starch intake (OR:1.01, 95% CI:1.00–1.01) and risk of MetS (p < 0.05). Similarly, subjects with an AA genotype and a high polyunsaturated fatty acid (PUFA) intake were at higher risk of MetS than those who were homozygous for the G allele. ConclusionsIndividuals with an AA genotype, and a high intake of MUFA, SFA, sugar and starch, had a high risk of developing MetS. Therefore, this genetic polymorphism might determine disease susceptibility when combined with a high consumption of fat and carbohydrate. A further study is necessary to confirm this association as a potential marker in predicting CVD events.

The expression of programmed death receptor 1 in non-small cell lung cancer and its clinicopathological features and prognosis showed a connection with epidermal growth factor receptor gene mutations

Objective: To investigate the relationships between the expression of programmed death 1 (PD-1) and the epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer (NSCLC). The study also attempted to investigate the clinicopathological features and prognosis in NSCLC patients. Methods: The expression of PD-1 protein in 88 cases of NSCLC tumor tissues and adjacent tissues was detected by immunohistochemistry. The mutations of EGFR in NSCLC were detected by Polymerase Chain Reaction-Amplification Refractory Mutation System(PCR-ARMS) method. The expression of PD-1 and patients' clinical characteristics and prognosis were analyzed. Results: PD-1 was positive in 63.6%(56/88) NSCLC tumor tissues, which was significantly higher than that in adjacent normal tissues (21.6%, 19/88) (P<0.05). EGFR gene mutations were found in 43 cases (48.9%), in which 30 cases (69.8%)were PD-1 positive expression. 45 cases had the wild types of EGFR gene, in which 26 cases (57.8%) were PD-1 positive. There were 24 cases of 19Del EGFR mutations, including 20 cases (83.3%) of PD-1 positive expression. 19 patients had 21L858 EGFR mutations, including 10 cases (52.6%) of PD-1 positive expression. The expression of PD-1 in NSCLC was related to patients' smoking status, lymph node metastasis and EGFR gene mutations (P<0.05). The median progression-free survival time of patients with PD-1 positive and negative expression was 7.03 and 18.66 months, respectively (P=0.007). In patients with wild-type EGFR gene, the median progression-free survival time of PD-1 positive and negative expression was 25.21 and 38.24 months, respectively. The difference was statistically significant (P=0.024). The median progression-free survival time in 43 cases of EGFR mutant patients with PD-1 positive and negative expression was 21.23 and 31.44 months. The difference was not statistically significant (P=0.128). Conclusions: PD-1 expresses in both EGFR mutant and wild-type NSCLC, and its expression levelis different with various EGFR mutations. The expression of PD-1 in NSCLC is related to the prognosis of patients, and the prognosis of patients with positive PD-1 expression was poor.

Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach.

Backgroundβ-Thalassemia is the most prevalent genetic disorder in India. Its traits and coinheritance vary from mild to severe conditions, resulting in thalassemia minor, intermediate, and major, depending upon many factors.PurposeThe objective of this study was to identify the incidence of β-thalassemia traits, their coinheritance, and mutations, as well as to support the patients already diagnosed with β-thalassemia in East-Western Indian population for better management.Patients and methodsSeventy-five referral cases for β-thalassemia were analyzed for various β-thalassemia traits, heterozygosity, and homozygosity conditions. Blood phenotypic parameters using cell counter and capillary electrophoresis were investigated. Analyses of eight common mutations of thalassemia in India were carried out using polymerase chain reaction-amplification refractory mutation system, end point polymerase chain reaction, and DNA sequencing methods.ResultsOf these (75) referral cases from East-Western Indian region, 68 were positive for β-thalassemia (90.67%). The majority of case types were of β-thalassemia minor (49, 65.33%), followed by HbE traits (6, 8.0%) and β-thalassemia major, including heterozygous and homozygous (5, 6.66%; 4, 5.33%) types and then HbE homozygous (2, 2.66%), as well as one each of the HbE/β-thalassemia and HbD/β-thalassemia (1, 1.34%) combination. Mutation analysis also revealed that the highest frequency of mutation was c.92+5G>C (41, 60.29%) followed by deletion 619bp (9, 13.23%) and c.79G>A (8, 11.76%) in our study group. Five cases (nos. 24, 27, 33, 58, and 71) exhibited coinheritance between β0/β+ (2), β0/β D (1), and c.124_127delTTCT/β+ or β0(2) affecting the Rajasthani and Gujarati populations in our study of the Western region of India.ConclusionWe strongly recommend these Western populations for genetic screening before adopting reproductive technologies and interracial marital relations.

Association of a Vascular Endothelial Growth Factor genetic variant with Serum VEGF level in subjects with Metabolic Syndrome

BackgroundThe metabolic syndrome (MetS) is a clustering of metabolic disorders that is associated with an increased risk of developing cardiovascular-disease, diabetes, and related diseases. Against this background, Vascular Endothelial Growth Factor (VEGF) plays an essential role in angiogenesis, vascular permeability, and hematopoiesis and its increased level is reported to be associated with increasing the risk of developing cardiovascular-disease, stroke and diabetes. Therefore the aim of present study was to explore the association of serum VEGF level and its associated genetic-polymorphism, rs10738760 (A>G) at 9p24.2, in 850 subjects with/without MetS. MethodsMetS was defined according to the International-Diabetes-Federation criteria. Genotyping was carried out using Polymerase chain reaction-amplification refractory mutation system. Anthropometric/biochemical parameters, including FBG, Triglyceride, HDL, TC, etc., were determined followed by univariate and multivariate analyses. ResultsMetS patients had significantly higher levels of BMI, waist-circumference, cholesterol, triglyceride, Hs-CRP and SBP/DBP, while the HDL-C levels was lower in patients group, compared to control group (P<0.05). Moreover, our analysis showed that MetS patients with GA or AA genotypes had a significantly (P=0.03) higher serum level of VEGF. Conclusionswe demonstrate an association between a VEGF genetic variant with MetS, suggesting its role as a risk stratification factor for MetS.

Diet Challenge in G6PD Deficient Egyptian Children: A One- Year Prospective Single Center Study with Genotype - Phenotype Correlation

▪Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent and add a burden on families in Egypt and Middle East due to lifelong diet restriction. Non-fava beans diet is the main food for most families in the region and parents and doctors consider it as a prohibited food whatever the genetic or clinical phenotype of G6PD. The effective management is avoiding a spectrum of food and drugs causing oxidative stress. No data is available about the hazards of consumption of non-fava beans diet.Aim: To investigate the effect of challenge of non-fava beans diet on occurrence of hemolysis in both common and rare mutations causing G6PD deficiency in Egyptian children as well as making a genotype-phenotype correlation.Patients and Methods: An interventional study registered in the Clinical Trials Government (NCT02498340) and included all G6PD deficient children who were regularly followed up in Pediatric Hematology Center, Ain Shams University over last decade from 2004-2014 who stopped eating non fava-bean diet since their diagnosis as G6PD deficient and willing to participate in the diet challenge. They were enrolled in a one year prospective study involved quantitative analyses for enzymatic activity, and molecular typing of G6PD enzyme using a polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique. Patient's medical records were reviewed as history of blood transfusion and G6PD level at diagnosis. Initial phase was dietetic challenge with ingestion of non-fava beans taken in small amount (10-20 gm/day for 3 successive days) for children with haemoglobin level ≥ 11 gm/dl with daily clinical and laboratory monitoring by complete blood count, and markers of hemolysis as well as measurement of MDA level both basal and at study end. A drop of Hb of 1.0 gm/dl and / or appearance of hemoglobinuria is considered a significant hemolysis. Patients who did not met the definition of significant hemolysis were prospectively followed up for one year with follow up during their chronic exposure by CBC and hemolysis markers/ 3 months.Results: 108 G6PD deficient patients were enrolled; their ages ranged between 1-12 year, (mean of 3.1±1.2) with a male to female ratio of 8:1. Genotypes were; Mediterranean variant in 53%, Cairo in 13% and African mutations in 16%. Rare mutations as Chatham in 4%, Santmaria in 1% and Asahi in 1%; were described in Egypt for the first time. As regard the initial clinical presentation, 17 (15.7%), were asymptomatic; (6 had Mediterranean variant (2 silent polymorphism), 2 the Cairo variant, one the Chatham variant, one in the Asahi variant and in seven patients the molecular variant was not identified. History of blood transfusion was reported in79% in Mediterranean variant, 61.5% in Cairo variant , 56% in African variant, 50% in Chatham variant, and in none of the santmaria and Asahi variants. However no significant relation was detected between mutations and classes of G6PD deficiency(severity) and also with blood transfusion requirement among studied cases. Neonatal jaundice was observed in 56.4% of studied patients with the highest percentage in Mediterranean variant. Clinical diversity showed 83% symptomatic; 64% received blood transfusion, history of ingestion of fava beans was reported in 70% while 61 % had history of neonatal jaundice. The G6PD enzyme level was significantly lowered in Mediterranean and African mutation compared to other mutations (P< 0.01) but it was not correlated with disease clinical severity. No hemolysis was reported after dietetic challenge in all different genotypes (no hemoglobinuria and absence of markers of hemolysis). No drop of Hb over a one year period of chronic consumption of non-fava beans diet. Moreover no significant difference was found between baseline hemoglobin and MDA and their levels at the study end (p>0.05).Conclusion: Chronic ingestion of small amount of non-fava beans once weekly for one year was not associated with haemolysis or increase in oxidative stress in this cohort of G6PD deficient children in all variants. Improved family satisfaction with 92% of patients accepted to continue food challenge with a great satisfaction. Mediterranean mutation was the most common variant. Three rare non-Middle-East mutations were reported for first time; namely Santamaria, chatham and Asahi. DisclosuresNo relevant conflicts of interest to declare.

Frequencies of the Common MEFV Gene Mutations in Adiyaman, Southeast Anatolia, Turkey.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by fever and serosal inflammation. The reasons for the disorder are mutations in the Mediterranean fever (MEFV) gene; the most common of which are M694V, M680I, M694I and V726A. In this study, we aimed to screen these common mutations of the MEFV gene and then determine the prevalence of FMF according to these mutations in Adıyaman, Southeast Anatolia, Turkey. Seven hundred and sixty-seven healthy individuals from the region of Adıyaman participated in the study. Polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) methods were used to determine the common mutations of the MEFV gene. Twenty-six (3.9%) individuals had only one mutation in the MEFV gene, 25 individuals were heterozygous and one person was homozygous for the V726A mutation (0.15%). In the present study, the V726A mutation (50.0%) was the most frequent, followed by M694V (38.5%), M680I (7.7%) and M694I (3.8%). It was seen that the carrier rate was very low and the prevalence of FMF was 0.15%, according to the common mutations of the MEFV gene in Adıyaman, Southeast Anatolia, Turkey.

Immunoregulatory cytokines gene polymorphisms in Egyptian patients affected with acquired aplastic anemia

The immune system is thought to play an important role in aplastic anemia (AA) in light of recent findings of hematologic reconstitution after immunosuppressive therapy. T cell activation, apoptosis, and the cytokines interferon- and TNF-α are suspected to play a role in the suppression of growth of progenitor cells and induced apoptosis in CD34 target cells, TGFβ is a multifunctional peptide, usually produced in latent form and requiring activation to produce a biological response. Also, TGF-β1 has been described as an important negative regulator of haemopoiesis. Over production of IL-6 is described in AA but is of unknown pathophysiological significance. To investigate the role of cytokine gene polymorphisms (IL-6/-174, TNF-α/-308, IFN-γ/+874, and TGFβ1/-509) in patients with acquired AA to assess if genotypes associated with higher or lower production were more prevalent than in established control population and to study the possible association of these genotypes with the disease severity. Fifty AA patients were included in this study. Polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique was used to detect INF-γ single nucleotide polymorphism -874A/T, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to assess IL-6-174 C/G, TNF-α-308G/A, and TGFb1-509C/T gene polymorphisms. Genotypes associated with high production of TNF-α, TGF-β and IFN-γ, and IL-6 were more frequent in patients than in control; no association was found between the presence of hypersecretory genotypes and the disease severity.

Prenatal diagnosis for thalassaemia in Egypt: what changed parents' attitude?

To present the current status of the prenatal diagnosis services and results from the largest thalassaemia center in Egypt treating 3000 patients. Traditionally, prenatal diagnosis has not been successful in reducing the births of affected children in Egypt, because the majority of women undergoing prenatal diagnosis continued to have affected pregnancies. Seventy-one pregnant mothers at risk for β-thalassaemia underwent prenatal diagnosis by chorionic villus sampling (n=57) or amniocentesis (n=14) between 11 to 14 weeks of gestation. Molecular characterization of fetal DNA by reverse dot blot hybridization and polymerase chain reaction-amplification refractory mutation system techniques was conducted in all cases. Twenty-four women (33.8%) were found to have affected fetuses; 100% of these women opted to terminate the pregnancy. The change in attitude towards termination of pregnancy was related to in-depth counseling of the religious aspects towards prenatal diagnosis and termination of pregnancy. Forty-eight women (66.2%) with normal or carrier fetuses for β-thal requested human leukocyte antigen typing of the fetal material to determine if the fetus was a human leukocyte antigen match for their existing thalassaemic siblings. This study demonstrates that prenatal diagnosis is feasible and acceptable in Egypt, a Muslim country, provided an in-depth discussion, which also addresses the religious considerations of prevention, is held with the couples.

Influence of cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in periodontitis

AimPersistent host inflammatory immune response against the pathogens results in the destruction of periodontal tissues. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a particularly important molecule in down-regulating T-cell expansion and cytokine production. This study aimed to assess three functional SNPs within CTLA-4 gene, −1722 T/C, −318 C/T, and +49 A/G in patients with aggressive or chronic periodontitis. Materials and methodsA total of 197 patients with periodontitis (71 aggressive and 126 chronic periodontitis) and 218 healthy controls were recruited. All samples were genotyped for CTLA-4 gene polymorphisms by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS). ResultsThe allelic and genotype frequencies of only +49 A/G SNP were more prominence in patients with chronic periodontitis (CP) than that controls (0.0005 and 0.001, respectively). Multivariate logistic regression analysis was demonstrated that homozygosity in +49 G/G had profoundly increased susceptibility for CP, OR=3.7 (95% CI; 1.6–8.5, P=0.001). In addition, comparison of CTLA-4 SNPs between patients with CP and aggressive periodontitis (AgP) revealed that heterozygosity in −1722 T/C polymorphism of CTLA-4 gene had a significantly higher risk for CP compared with AgP with a calculated odds ratio of 2.18 (95% CI; 1.17–4.06, P=0.01). ConclusionThese results suggest that CTLA-4 gene variants might be associated to susceptibility to specific form of periodontitis and participate in the CP development.

Molecular Basis of β-Thalassemia in the Western Province of Saudi Arabia: Identification of Rare β-Thalassemia Mutations

This study aimed at the identification of the spectrum of mutations in patients with β-thalassemia (β-thal) in the western province of Saudi Arabia. Screening for the mutations was done using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique to test for 12 mutations, and direct automated DNA sequencing for the unknown samples. The study included 172 patients; of these 15 patients had sickle cell anemia and one Hb S [β6(A3)Glu→Val, GAG>GTG]/β-thal. A total of 23 mutations were identified to cause the disease in the western area. Seven common mutations were responsible for the β-thal alleles in 78% of patients and could be detected by the ARMS technique: IVS-II-1 (G>A), IVS-I-110 (G>A), IVS-I-5 (G>C), codon 39 (C>T), codon 26 (G>A) [Hb E or β26(B8)Glu→Lys, GAG>AAG], frameshift codons (FSC) 8/9 (+G), and IVS-I-1 (G>A). DNA sequencing of uncharacterized alleles detected eight less common mutations: FSC 41/42 (–TCTT), IVS-I 25 bp deletion, codon 37 (G>A), FSC 44 (–C), Cap site +1 (A>C), IVS-I-6 (T>C), FSC 5 (–CT) and IVS-I-1 (G>T), and eight rare mutations: −87 (C>G), initiation codon −1 (T>G), codon 15 (G>A), FSC 16 (–C), FSC 20/21 (+G), codon 27 (G>A), IVS-I-130 (G>C) and IVS-II-837 (A>C). Four alleles were normal by DNA sequencing. Genetic heterogeneity was observed in this study, 10 mutations were of Asian or Asian/Indian origin, two were Kurdish, one Chinese, one Turkish, one Saudi, and the remainder were of Mediterranean origin. The presence of a large population of immigrants in the western province is responsible for the great heterogeneity at the molecular level, and for the difference observed in the frequencies of mutations from those reported in the eastern province of Saudi Arabia. Screening for β-thal mutations using PCR-ARMS for the seven most frequent mutations in the Saudi population followed by DNA sequencing of the unknown alleles could be useful for the implementation of a strategy for carrier detection and preimplantation genetic diagnosis in high risk families.

β-Thalassemia Mutations Found During 1 Year of Prenatal Diagnoses in Fars Province, Iran

We report the spectrum of β-thalassemia (β-thal) mutations observed in a cohort of at-risk couples, who presented for prenatal diagnosis at the Thalassemia, Hemophilia and Prenatal Diagnosis Genetic Research Center, Shiraz Medical University, Fars, Iran, from March 2001 to April 2002. Using polymerase chain reaction-amplification refractory mutation system (PCR-ARMS), restriction fragment length polymorphism (RFLP) and direct sequencing technologies, in different combinations, we were able to provide preventive medical abortions to 55 couples at-risk of having an affected fetus. Fetal samples of chorionic villus (63.6%) or amniocentesis (37.6%) were collected according to the gestational age. The average presentation age was 11.6 ± 2.6 weeks. The expected prevalent mutations were IVS-II-1 (G>A, 23.6%) and IVS-I-110 (G>A, 10.0%) followed by IVS-I-5 (G>C, 6.4%) and IVS-I, 25 bp deletion (8.2%).

Incidence and Association of 563 C/T Mediterranean and the Silent 1311C/T G6PD Mutations in G6PD-deficient Egyptian Children

Background: Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency variants is essential, especially since the biochemical characterization has lost its significance due to the individual variability. As a result, cases can be misdiagnosed. The present study was designed to determine the incidence of G6PD Mediterranean (Med) mutation among Egyptian children with G6PD deficiency as well as its molecular association with the G6PD 1311T silent polymorphism. Methods: Fifty G6PD-deficient children were subjected to quantitative G6PD enzyme assay. A polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique was used to detect the G6PD Med mutation, and polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) was used to assess the associated G6PD 1311T silent polymorphism. Results: G6PD Med was found in 62% and G6PD 1311T polymorphism in 52% of the patients. The association between both mutations was statistically significant. Conclusion: Glucose-6-phosphate dehydrogenase Med mutation is 1 of the most common mutations causing G6PD deficiency among Egyptian children with G6PD deficiency, with linkage disequilibrium between this G6PD mutation and thymine at nucleotide (nt) 1311.

Detection of responsible mutations for beta thalassemia in the Kermanshah Province of Iran using PCR-based techniques

Beta Thalassemia has been reported to be a common genetic disorder in Iran. To establish the molecular spectrum of the beta thalassemias in the Kermanshah Province of Iran, 185 unrelated beta thalassemia patients with Kurdish ethnic background were studied (181 beta-thalassemia major and 4 beta-thalassemia intermedia). Using polymerase chain reaction-amplification refractory mutation system (PCR-ARMS), restriction fragment length polymorphism (RFLP) and direct genomic sequencing twenty different mutations were identified accounting for 98.1% of the alleles. Approximately 80.8% of beta-thalassemia chromosomes had beta(0) mutation. The most prevalent mutation was the IVSII-1 (G-->A) (32.97%), followed by CD8/9 +G (13.51%), IVSI-110 (C-->T) (8.38%), CD 36/37 -T (7.84%), CD8 -AA (5.94%), CD15 (G-->A) (4.86%) and IVSI-1 (G-->A) (4.59%). All of these mutations accounted for 78.1% of the alleles. The results described here will be of valuable help in the development of successful prevention programs for the population of Kermanshah.

Is the Frameshift Codons 8/9 (+G) [FSC 8/9 (+G)] β-Thalassemia Mutation, Detected by the Polymerase Chain Reaction-Amplification Refractory Mutation System, Really FSC 8/9 (+G)?

There are several polymerase chain reaction (PCR)-based approaches for the analysis of known mutations. The PCR-amplification refractory mutation system (PCR-ARMS) is one of the best known and frequently used for the detection of β-thalassemia (β-thal) mutations. However, there is an important point to be considered when searching for the frameshift codon (FSC) 8 (–AA) and FSC 8/9 (+G) mutations. Whereas the primer is specific for the FSC 8 mutation only, the primer for the FSC 8/9 mutation does not discriminate between the FSC 8/9 and FSC 8 mutations. Thus, the high number of FSC8/9 mutations reported in countries like India, Pakistan and some regions of Iran may be due to the use of the FSC 8/9 primer, without taking the FSC 8 mutation into account. It is thus advisable to test for FSC 8 before the FSC 8/9 mutation whenever PCR-ARMS is the method of investigation for these two mutations.

β-Thalassemia Mutations in the Iranian Kurdish Population of Kurdistan and West Azerbaijan Provinces

The aim of this study was to investigate the prevalence and spectrum of β-thalassemia (β-thal) mutations in the population of Sunni Muslim Kurds in western Iran and to set up a prenatal diagnostic laboratory. Sixty unrelated Kurdish β-thal patients identified in hematology clinics from different cities were studied. The mutations in 120 chromosomes were studied by polymerase chain reaction–amplification refractory mutation system and direct sequencing methods. We found fifteen β-thal mutations, and IVS-II-1 (G>A) was the most frequent, comprising 35% of all mutations. Other common mutations were frameshift codons 8/9 (+G) 15.7%, IVS-I-1 (G>A) 8%, FSC 5 (–CT) 6.7%, FSC 8 (–AA) 6.7%, and IVS-I-110 (G>A) 6%. This is the first comprehensive study in this region and could provide a reference for prenatal testing and genetic counseling in this population.

Fibrogenic Polymorphisms (TGF-β, PAI-1, AT) in Mexican Patients With Established Liver Fibrosis. Potential Correlation With Pirfenidone Treatment

Background/AimThe aim of this work was to establish a potential correlation between specific polymorphisms and presence of hepatic fibrosis in Mexican patients with established liver fibrosis (ELF). Second, necroinflammatory index...

Molecular Spectrum of β-Thalassemia Mutations in Northwestern Iran

β-Thalassemia (β-thal) is a hereditary autosomal disorder with decreased or absent β-globin chain synthesis. This study was designed to identify the common and rare β-thal mutations in the Azerbaijan provinces, Northwestern Iran, and to set up a prenatal diagnostic laboratory. One hundred unrelated patients with known β-thal major and intermedia, registered with the thalassemia clinics in the provincial capitals of Tabriz and Ardebil, were included. Mutations were studied in 200 chromosomes, by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) and direct sequencing methods. We found 17 β-thal mutations in this region of Iran. The results showed that IVS-II-1 (G→A) was the most frequent mutation, comprising 21% of all mutations. Other common mutations were IVS-I-110 (G→A) 18%, frameshift codons (FSC) 8/9 (+G) 14.5%, FSC 8 (−AA) 8% and IVS-I-1 (G→A) 7.5%. This is the first comprehensive study in this region and could be useful for developing a β-thal molecular screening in Azerbaijan-Iran

Study of β-Thalassemia Mutations Using the Polymerase Chain Reaction-Amplification Refractory Mutation System and Direct DNA Sequencing Techniques in a Group of Egyptian Thalassemia Patients

The aim of this study was the molecular characterization of β-thalassemia (thal) mutations in a group of 95 Egyptian thalassemic patients from Fayoum in Upper Egypt, Cairo, Alexandria and Tanta in Lower Egypt and the Nile Delta. To identify these anomalies, the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique was used, complemented by direct DNA sequencing for uncharacterized cases.In 80 of the 95 patients, the β-thal mutation was detected by PCR-ARMS. The most common allele encountered in our study was IVS-I-6 (T→C) (36.3%); the second most common mutation was IVS-I-110 (G→A) (25.8%). In addition, we report three homozygous cases for the promoter region −87 (C→G) allele with a frequency of 3.2%. DNA sequencing of uncharacterized cases (14 cases, 15 alleles) revealed six cases (six alleles) of codon 27 (G→T), and three cases (three alleles) of the IVS-II-848 (C→A) mutation. Codon 37 (G→A) in the homozygous state was found in one patient with positive consanguinity. The frameshift codon 5 (−CT) mutation was detected in two of our uncharacterized cases. The codon 15 (TGG→TGA) mutations was detected in one patient (one allele, 0.5%). All studied cases were fully characterized by this strategy.Screening for β-thalassemic mutations using ARMS-PCR for the seven most frequent alleles in Egypt succeeded in determining the β-globin genotype in 84.2% of our patients (91.6% of the expected alleles). To improve the efficiency of routine screening, the PCR-ARMS mutation panel should be updated to include the reported rare alleles. Direct DNA sequencing is an additional way to allow a full characterization of β-thal patients in the Egyptian population.