A phased SNP-based classification of sickle cell anemia HBB haplotypes

Background: Sickle cell anemia causes severe complications and premature death. Five common beta-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the beta-globin gene cluster. This is labor intensive, and error prone. Methods: We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles. Results: We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs (rs3834466, rs28440105, rs10128556, and rs968857) marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs. Conclusion: Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources.NIH Bethesda, MDBoston Univ, Sch Med, Dept Med, 72 E Concord St, Boston, MA 02118 USABoston Univ, Bioinformat Program, Boston, MA 02215 USAKing Saud Univ, Coll Med, Sickle Cell Dis Res Ctr, Riyadh, Saudi ArabiaKing Saud Univ, Coll Med, Dept Pediat, Riyadh, Saudi ArabiaKing Faisal Univ, Al Omran Sci Chair, Al Hasa, Saudi ArabiaImam Abdulrahman bin Faisal Univ, Inst Res & Med Consultat, Dammam, Saudi ArabiaEscola Paulista Med, Hematol & Blood Transfus Div, São Paulo, BrazilBoston Univ, Sch Publ Hlth, Dept Biostat, Boston, MA 02118 USAEscola Paulista Med, Hematol & Blood Transfus Div, São Paulo, BrazilNIH: R01 HL 068970NIH: RC2 HL 101212NIH: R01 87681NIH: T32 HL007501Web of Scienc

Bedside testing of CYP2C19 gene for treatment of patients with PCI with antiplatelet therapy

BACKGROUND: To mitigate the risk of stent thrombosis, patients treated by percutaneous coronary intervention (PCI) are administered dual anti-platelet therapy comprising aspirin and a platelet P2Y12 receptor inhibitor. Clopidogrel is a prodrug requiring activation by the cytochrome P450 enzyme, CYP2C19. In Saudi Arabia, it has been reported that approximately 26% of the population carries CYP2C19*2 and/or *3 loss-of-function polymorphisms in addition to a high prevalence of CVD. METHODS: This prospective (April 2013-December 2020) parallel assignment clinical trial focuses on ST-Elevation Myocardial Infarction (STEMI) patient outcomes. The clinical trial includes 1500 STEMI patients from two hospitals in the Eastern Province of Saudi Arabia. Patients are assigned to one of two groups; the control arm receives conventional therapy with clopidogrel, while in the active arm the Spartan RX CYP2C19 assay is used to determine the *2 genotype. Carriers of a CYP2C19*2 loss-of-function allele receive prasugrel or ticagrelor, while non-carriers are treated with clopidogrel. Follow-up is one year after primary PCI. The primary end point is the number of patients who develop an adverse major cardiovascular event, including recurrent MI, non-fatal stroke, cardiovascular death, or major bleeding one year after PCI. DISCUSSION: The risk of stent thrombosis in PCI patients is usually reduced by dual anti-platelet therapy, comprising aspirin and a P2Y12 inhibitor, such as clopidogrel. However, clopidogrel requires activation by the cytochrome P450 enzyme, CYP2C19. Approximately 20% of the population are unable to activate clopidogrel as they possess the CYP2C19*2 loss-of function (LoF) allele. The primary goal of this trial is to study the benefits of treating only those patients that cannot activate clopidogrel with an alternative that has shown to be a more effective platelet inhibitor and does not require bioactivation by the cytochrome P450 enzyme. We expect an improvement in net clinical benefit outcome in the active arm patients, thus supporting pharmacogenetic testing in PCI patients post STEMI. TRIAL REGISTRATION: Trial registration name is "Bedside Testing of CYP2C19 Gene for Treatment of Patients with PCI with Antiplatelet Therapy" (number NCT01823185) retrospectively registered with clinicaltrials.gov on April 4, 2013. This trial is currently at the patient recruitment stage

Comparative and molecular analysis of MRSA isolates from infection sites and carrier colonization sites

Abstract Background Methicillin resistant Staphylococcus aureus (MRSA) constitutes a major global health concern causing hospital and community acquired infections. A wide diversity of MRSA genotypes are circulating in geographically related regions. Therefore understanding the molecular epidemiology of MRSA is fundamental to design control and clearance measures. Methods A total of 106 MRSA isolates from infection (51) and carrier colonization sites (55) are characterized genetically based on SCCmec and MLST genotyping methods in addition to detection of PVL, TSST-1 and enterotoxins. Results Sccmec-IV was the most frequently detected genotype (77.3%) followed by genotype V (13.2%) and III (9.4%). SCCmec-IVa was more prevalent among the carrier group (p value 0.002). CC80 was the most commonly identified clonal complex (CC). CC6 and CC22 were significantly more prevalent among the carrier group (p value 0.02 and 0.01, respectively). PVL was highly prevalent among the isolates (58.5%). PVL was detected in 70.6% of isolates from infection sites and 47.3% of isolates from carriers. All strains were sensitive to vancomycin, however, MRSA strains isolated from infection sites had significantly higher MICs compared to strains isolated from carrier colonization sites (p value 0.021). Five new sequence types mainly from the carrier group were identified and described in the study. Conclusions MRSA population is genetically very diverse among carriers and infected individuals. With SCCmec type IV being most prevalent, this suggests a community origin of most MRSA strains. Therefore very well designed surveillance and clearance strategies should be prepared to prevent emergence and control spread of MRSA in the community

Molecular Bases of β-Thalassemia in the Eastern Province of Saudi Arabia

β-thalassemia is a group of heterogeneous recessive disorders common in many parts of the world. Al-Qatif and Al-Hassa oases in the Eastern Province of Saudi Arabia are regions known for high frequency of these disorders. Using two molecular methods, based on multiplexing-amplification refractory system and reverse hybridization principles, the spectrum of β-thalassemia in the region was studied. Sixty-nine subjects with known β-thalassemia disease and volunteers with high hemoglobin A2(HbA2) and low mean corpuscular volume (MCV) were included in this study. Ten mutations were detected in 91% of the subjects under study. Six of these mutations had previously been observed while the other four mutations are reported here for the first time. In addition, four of the mutations accounted for 76.8% of the subjects studied. IVSII-1 (G > A), IVSI-5 (G > A), and codon 39 (C > T) mutations were found to be the most frequent. However, the frequencies of different mutations reported here are slightly different from those reported earlier. A number of these mutations were also found in the neighboring countries, which can be explained in terms of gene flow

Intronic Polymorphisms in the CDKN2B-AS1 Gene Are Strongly Associated with the Risk of Myocardial Infarction and Coronary Artery Disease in the Saudi Population

Recent genome-wide association studies identified single nucleotide polymorphisms (SNPs) on the chromosome 9p21.3 conferring the risk for CAD (coronary artery disease) in individuals of Caucasian ancestry. We performed a genetic association study to investigate the effect of 12 candidate SNPs within 9p21.3 locus on the risk of CAD in the Saudi population of the Eastern Province of Saudi Arabia. A total of 250 Saudi CAD patients who had experienced an myocardial infarction (MI) and 252 Saudi age-matched healthy controls were genotyped using TaqMan assay. Controls with evidenced lack of CAD provided 90% of statistical power at the type I error rate of 0.05. Five percent of the results were rechecked for quality control using Sanger sequencing, the results of which concurred with the TaqMan genotyping results. Association analysis of 12 SNPs indicated a significant difference in the genotype distribution for four SNPs between cases and controls (rs564398 p = 0.0315, χ2 = 4.6, odds ratio (OD) = 1.5; rs4977574 p = 0.0336, χ2 = 4.5, OD = 1.4; rs2891168 p = 1.85 × 10 − 10, χ2 = 40.6, OD = 2.1 and rs1333042 p = 5.14 × 10 − 9, χ2 = 34.1, OD = 2.2). The study identified three protective haplotypes (TAAG p = 1.00 × 10 − 4; AGTA p = 0.022 and GGGCC p = 0.0175) and a risk haplotype (TGGA p = 2.86 × 10 − 10) for the development of CAD. This study is in line with others that indicated that the SNPs located in the intronic region of the CDKN2B-AS1 gene are associated with CAD