title: Genotyping of BCL11A and HBS1L-MYB SNPs associated with fetal haemoglobin levels: a SNaPshot minisequencing approach.
abstract: BACKGROUND
  B-thalassaemia and sickle cell disease (SCD) are two of the most common monogenic diseases that are found in many populations worldwide. In both disorders the clinical severity is highly variable, with the persistence of fetal haemoglobin (HbF) being one of the major ameliorating factors. HbF levels are affected by, amongst other factors, single nucleotide polymorphisms (SNPs) at the BCL11A gene and the HBS1L-MYB intergenic region, which are located outside the β-globin locus. For this reason, we developed two multiplex assays that allow the genotyping of SNPs at these two genomic regions which have been shown to be associated with variable HbF levels in different populations.
  
  
  RESULTS
  Two multiplex assays based on the SNaPshot minisequencing approach were developed. The two assays can be used to simultaneous genotype twelve SNPs at the BCL11A gene and sixteen SNPs at HBS1L-MYB intergenic region which were shown to modify HbF levels. The different genotypes can be determined based on the position and the fluorescent colour of the peaks in a single electropherogram. DNA sequencing and restriction fragment length polymorphism (PCR-RFLP) assays were used to verify genotyping results obtained by SNaPshot minisequencing.
  
  
  CONCLUSIONS
  In summary, we propose two multiplex assays based on the SNaPshot minisequencing approach for the simultaneous identification of SNPs located at the BCL11A gene and HBS1L-MYB intergenic region which have an effect on HbF levels. The assays can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.
authors: Pavlos
authors: P
authors: Fanis
authors: Ioanna
authors: I
authors: Kousiappa
authors: Marios
authors: M
authors: Phylactides
authors: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 International Airport Avenue, Agios Dometios, Nicosia 1683, Cyprus. marinakl@cing.ac.cy.
authors: Marina
authors: M
authors: Kleanthous
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D021901:DNA, Intergenic / Q000235:genetics* / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000032:analysis / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics; D005838:Genotype; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C486563:BCL11A protein, human; D002352:Carrier Proteins; D021901:DNA, Intergenic; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D010445:Peptide Elongation Factors; D020598:Proto-Oncogene Proteins c-myb; D012097:Repressor Proteins; D005319:Fetal Hemoglobin; D019204:GTP-Binding Proteins; C000628258:HBS1L protein, human