Reaction-amplification Refractory Mutation System Research Articles

Molecular basis of genetic improvement for key rice quality traits in Southern China

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

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.

Genotyping of blood groups in alloimmunized patients with β-thalassemia major by T-ARMS-PCR and multiplex-aso-pcr

IntroductionBeta-thalassemia major is a severe hemolytic anemia requiring life-long blood transfusion. Planned random donor blood transfusion is associated with alloimmunization against incompatible antigens. Determination of the minor blood group systems phenotype or genotype, and administration of the compatible blood components can significantly reduce the rate of alloimmunization.The present study aimed to determine the prevalence of alloimmunization, and genotype/phenotype characteristics of the minor blood groups systems in patients with β-thalassemia major. Material and methodsThis study was conducted on 1147 β-thalassemia major patients. Initially, antibody screening and antibody identification were performed. Then, phenotyping and genotyping for the Rh, Kell, Kidd, and Duffy blood groups were done in alloimmunized patients using monoclonal antibodies and Multiplex-Allele Specific Oligonucleotide-Polymerase Chain Reaction (Multiplex-ASO-PCR) and Tetra-primer amplification refractory mutation system–PCR (T-ARMS-PCR), respectively. Any phenotype/genotype discrepancy was assessed by direct sequencing. ResultsNinety-seven (8.5 %) out of 1147 patients had alloantibodies against the minor blood group antigens (44 males, 45.4 %, and 53 female, 54.6 %). The most common alloantibodies were against the RH (n: 47, 48.5 %), and the Kell (n: 23, 23.7 %) blood groups systems. Twenty-three (2.1 %) genotype/phenotype discrepancies out of 1067 tests, including 9 in the Rh (9.3 %), 8 in Duffy (34.8 %), and 6 in Kidd (26.1 %) blood groups were detected. No discrepancy was found in the Kell blood group system. Direct sequencing revealed that the results of molecular methods were correct. ConclusionMultiplex-ASO-PCR and T-ARMS-PCR molecular methods are fast, reliable and cost-benefit molecular methods for the minor blood group genotyping in multi-transfused β-thalassemia major patients.

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.

Iranian patients with hemoglobin H disease: genotype-phenotype correlation.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

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.

The TLR2 and TLR4 gene polymorphisms in Moroccan visceral leishmaniasis patients

Visceral leishmaniasis (VL) is endemic in the Mediterranean basin and leads to the most severe form of Leishmania infection, lethal if left untreated. However, most infections are sub-clinical or asymptomatic, reflecting the influence of host genetic background on disease outcome. This study aimed to investigate possible association of TLR4 Asp299Gly, TLR4 Thr399Ile and TLR2 Arg753Gln polymorphisms with VL in Moroccan children. We enrolled 119 children with VL caused by Leishmania infantum as well as 138 unrelated children, 95 asymptomatic subjects and 43 healthy individuals who had no evidence of present or past infection. Polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). Results showed significant differences in genotype Thr399Ile and recessive model frequencies between VL and delayed-type hypersensitivity (DTH+) groups (p=0.018, OR=0.414CI 0.195–0.880; p=0.029, OR=0.448CI 0.214–0.938], respectively) by having the amino-acid threonine polymorphism as a reference in the VL group. Concerning the Asp299Gly there were a significant associations when comparing VL vs DTH+ (Asp299Gly genotype p=0.002, OR=0.326CI 0.158–0.671, allele frequencies p=0.033, OR=0.396CI 0.164–0.959, recessive model p=0.002, OR=0.343CI 0.172–0.681) and DTH+ vs DTH- groups (Asp299Gly genotype p=2.160E-4, OR=3.065CI 1.672–5.618, Gly299Gly genotype p=0.047, OR=0.368CI 0.299–0.452, allele frequencies p=1.406E-7, OR=29.571CI 3.907–223.8, recessive model p=4.370E-14, OR=36.965CI 8.629–158.3), by having the aspartic acid polymorphism as a reference these results suggest that the allele A (savage) confer protection against the clinical manifestations but not against the infection. Furthermore, there was a significant association regarding the Arg753Gln genotype (p=0.002, OR=0.326CI 0.158–0.671), allele frequencies (p=0.033, OR=0.396CI 0.164–0.959) and when applying a recessive model (p=0.002, OR=0.343CI 0.172–0.681) in the VL vs DTH+ groups. The same results was observed when comparing DTH+ vs DTH- groups (p=4.136E-6, OR=0.211CI 0.104–0.428), allele frequencies (p=0.008, OR=0.327CI 0.137–0.779) and recessive model (p=1.748E-5, OR=0.244CI 0.124–0.480). The results provide evidence that allele C in Thr399Ile and allele G in Arg753Gln polymorphisms may lead to protection against the clinical disease. Our data provide insights into the possible role of TLR2 and TLR4 variations in VL susceptibility.

A sensitive and practical method to detect the T790M mutation in the epidermal growth factor receptor.

The current study aimed to develop a method to rapidly, sensitively and practically screen for the epidermal growth factor receptor (EGFR) T790M mutation. This method combines an allele-specific competitive blocker (ACB) with a TaqMan quantitative polymerase chain reaction (PCR) amplification refractory mutation system (ARMS) in a one-step reaction. Using a mimic of a human genomic DNA panel containing serially diluted mutant alleles, the performance efficacy of this method was assessed. Using this method, the EGFR T790M mutation was detected in tyrosine kinase inhibitor (TKI)-naïve samples obtained from 27 non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. The association between de novo T790M mutations and the clinical benefit of EGFR-TKI treatment was also analysed. The sensitivity of this method was as low as 0.01%. In the samples from the 27 NSCLC patients, this method identified 6 mutant patients (22.2%), which was higher than the detection rate with scorpion ARMS (0.0%). No clinical variables were associated with the occurrence of a de novo T790M mutation. The median progression-free survival time in the TKI-naïve patients with a T790M mutation was shorter that that of patients without the mutation, but the difference was not significant (3.2 vs. 19.5 months, respectively; P=0.256). The median overall survival time in the groups with or without T790M mutation also did not significantly differ (10 vs. 20 months, respectively; P=0.689). Overall, the ACB-ARMS PCR method could be useful for detecting the EGFR T790M mutation in clinical samples that contain only a small number of mutant alleles. The clinical significance of a de novo T790M mutation should be further investigated.

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.

Association between polymorphisms of PSMB8, PSMB9 and TAP2 genes with rheumatoid arthritis in ethnic Han Chinese from Yunnan

To assess the association between single nucleotide polymorphisms (SNPs) of PSMB8, PSMB9 and TAP2 genes and rheumatoid arthritis (RA) in ethnic Han Chinese from Yunnan.A case-control study was carried out using 177 RA patients and 288 healthy controls. Genotypes of rs2071543, rs55745125 and rs138635403 loci of PSMB8 gene, and rs17587 locus of PSMB9 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). And a polymerase chain reaction amplification refractory mutation system (ARMS-PCR) was used for typing rs2228396 locus of TAP2 gene. Genotypic and allelic frequencies were calculated. An Epi Info 7 software was used to calculate the Odds Ratio (OR) of above SNPs between the two groups.Allelic and genotypic frequencies of rs138635403 and rs17587 loci have differed significantly between the two groups (P<0.05). The frequency of GG genotype for rs17587 locus was also higher in the RA group (0.672) compared with control group (0.524) (OR=1.862, 95%CI: 1.261-2.749).Genetic polymorphisms of rs17587 appeared to be associated with RA in ethnic Han Chinese from Yunnan.

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.