Abstract
BackgroundB-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.ResultsTwo 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.ConclusionsIn 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.
Highlights
B-thalassaemia and sickle cell disease (SCD) are two of the most common monogenic diseases that are found in many populations worldwide
We report the development of two multiplex assays based on the SNaPshot-minisequencing approach for simultaneous screening of twelve and sixteen known single nucleotide polymorphisms (SNPs), which have an effect on HbF levels in different populations, in the BCL11A gene and HBS1L-MYB intergenic region, respectively
The data suggest that these polymorphisms are associated with variable HbF levels in different ethnic populations with hereditary persistence of fetal haemoglobin (HPFH), β-thalassaemia, sickle cell anaemia, β-thalassaemia heterozygotes and HbE heterozygotes
Summary
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. HbF levels are affected by genetic factors outside of the β-globin gene cluster
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