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We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs, UGT1A1⁎6 (rs4148323) and UGT1A1⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including UGT1A1⁎6 and UGT1A1⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples. [ABSTRACT FROM AUTHOR]

Clopidogrel is an antiplatelet drug especially used in patients undergoing percutaneous coronary interventions (PCI). Polymorphisms within CYP2C19 can result in important interindividual variations regarding therapeutic efficacy. Therefore, we aimed to evaluate the impact of the CYP2C19⁎2 variant (rs4244285) on in-stent restenosis occurrence in Chilean patients who underwent PCI and received clopidogrel. A total of 77 cases with stenosis >50% in the angioplasty site (62.75 ± 9.8 years, 80.5% males) and 86 controls (65.45 ± 9.8 years, 72.1% males) were studied. The polymorphism was genotyped using TaqMan® Drug Metabolism Genotyping Assays. Overall, CYP2C19⁎2 allele frequency was 8.3%. Diabetes, chronic lesions, and bare metal stents (BMS) were observed more often in cases than in controls (p = 0.05, p = 0.04, and p = 0.02, resp.). Genotypic frequencies did not differ significantly between the groups (p = 0.15). Nonetheless, the mutated allele was observed in a greater proportion in patients without in-stent restenosis (p = 0.055). There was no significant association between the rs4244285 variant and the occurrence of in-stent restenosis after PCI (OR = 0.44; 95% CI: 0.19 to 1.04; p = 0.06). In summary, no association was identified between the CYP2C19⁎2 variant and the development of coronary in-stent restenosis. [ABSTRACT FROM AUTHOR]

Background and Objectives. β-Thalassemia and sickle cell disease are genetic disorders characterized by reduced and abnormal β-globin chain production, respectively. The elevation of fetal hemoglobin (HbF) can ameliorate the severity of these disorders. In sickle cell disease patients, the HbF level elevation is associated with three quantitative trait loci (QTLs), BCL11A, HBG2 promoter, and HBS1L-MYB intergenic region. This study elucidates the existence of the variants in these three QTLs to determine their association with HbF levels of transfusion-dependent Saudi β-thalassemia patients. Materials and Methods. A total of 174 transfusion-dependent β-thalassemia patients and 164 healthy controls from Eastern Province of Saudi Arabia were genotyped for fourteen single nucleotide polymorphisms (SNPs) from the three QTL regions using TaqMan assay on real-time PCR. Results. Genotype analysis revealed that six alleles of HBS1L-MYB QTL (rs9376090C p=0.0009, rs9399137C p=0.008, rs4895441G p=0.004, rs9389269C p=0.008, rs9402686A p=0.008, and rs9494142C p=0.002) were predominantly associated with β-thalassemia. In addition, haplotype analysis revealed that haplotypes of HBS1L-MYB (GCCGCAC p=0.022) and HBG2 (GTT p=0.009) were also predominantly associated with β-thalassemia. Furthermore, the HBS1L-MYB region also exhibited association with the high HbF cohort. Conclusion. The stimulation of HbF gene expression may provide alternative therapies for the amelioration of the disease severity of β-thalassemia. [ABSTRACT FROM AUTHOR]

Tobacco smoking continues to be a leading cause of disease and mortality. Recent research has confirmed the important role of nicotinic acetylcholine receptor (nAChR) gene cluster on chromosome 15q 24-25 in nicotine dependence and smoking. In this study we tested the association of smoking initiation, age at onset of daily smoking, and heaviness of smoking with five single nucleotide polymorphisms (SNPs) within the CHRNA5-CHRNA3-CHRNB4 cluster. The group of 389 adult subjects of European ancestry from the north of Poland, including 212 ever (140 current and 72 former) and 177 never smokers with mean age 49.26, was genotyped for rs16969868, rs1051730, rs588765, rs6495308, and rs578776 polymorphisms. Distributions of genotypes for rs16969868 and rs1051730 were identical so they were analyzed together. Further analysis revealed the association between rs16969868-1051730 (OR = 2.66; 95% CI: 1.30–5.42) and number of cigarettes smoked per day (CPD) with heaviness of nicotine addiction measured by the Fagerström Test for Nicotine Dependence (FTND) (OR = 2.60; 95% CI: 1.24–5.43). No association between these polymorphisms and other phenotypes was found. Similarly, the association between rs588765, rs6495308, rs578776, and analyzed phenotypes was not confirmed. This study provides strong evidence for the role of the CHRNA5-CHRNA3-CHRNB4 cluster in heaviness of nicotine addiction. [ABSTRACT FROM AUTHOR]