DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease.
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ABSTRACT: Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620-1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of (G)gamma-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 x 10(-42)). Together, common SNPs at the BCL11A, HBS1L-MYB, and beta-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.
Project description:Sickle Cell Anemia (SCA) is one of the most common monogenic disorders worldwide. Molecular modifiers of clinical symptoms play an essential role in the amelioration of the effects of the disease. Single Nucleotide Polymorphisms (SNPs) of the BCL11A gene and within the HBS1L-MYB intergenic region, which are located outside the β-globin locus on chromosome 11, are considered to be genetic modifiers that are associated with elevated levels of foetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin, HbS. The work reported here aimed to detect the most common SNPs of BCL11A and HBS1L-MYB related to HbF in SCA patients and to estimate the frequency of occurrence of these genotypes. A total of 132 SCA patients whose condition was stable were recruited from Jeddah city, Saudi Arabia. SNPs at site locus rs4671393 on BCL11A, and at loci rs28384513 and rs9399137 on HBS1L-MYB were identified using TaqMan genotyping assay. Haematological parameters were analysed based on complete blood count and haemoglobin separation using the capillary electrophoresis technique. Highly significant differences in the diagnostic haematological parameters, including all blood-cell types and HbF, were observed between the study cohort and control groups. We also found that BCL11A rs4671393 genotypes of GG and AG were more likely to show increases in HbF levels than other genotypes. In addition, a strong relationship was found between HBS1L-MYB rs9399137 and rs28384513 genotypes in the cohort, whereas no significant association was observed between BCL11A rs4671393 variant and other variants. Our study highlights the importance of investigating genetic determinants that play roles in the amelioration of the severity of clinical symptoms and complications of SCA.
Project description:BackgroundGenetic variation at loci influencing adult levels of HbF have been shown to modify the clinical course of sickle cell disease (SCD). Data on this important aspect of SCD have not yet been reported from West Africa. We investigated the relationship between HbF levels and the relevant genetic loci in 610 patients with SCD (98% HbSS homozygotes) from Cameroon, and compared the results to a well-characterized African-American cohort.Methods and findingsSocio-demographic and clinical features were collected and medical records reviewed. Only patients >5 years old, who had not received a blood transfusion or treatment with hydroxyurea were included. Hemoglobin electrophoresis and a full blood count were conducted upon arrival at the hospital. RFLP-PCR was used to describe the HBB gene haplotypes. SNaPshot PCR, Capillary electrophoresis and cycle sequencing were used for the genotyping of 10 selected SNPs. Genetic analysis was performed with PLINK software and statistical models in the statistical package R. Allele frequencies of relevant variants at BCL11A were similar to those detected in African Americans; although the relationships with Hb F were significant (p <.001), they explained substantially less of the variance in HbF than was observed among African Americans (∼ 2% vs 10%). SNPs in HBS1L-MYB region (HMIP) likewise had a significant impact on HbF, however, we did not find an association between HbF and the variations in HBB cluster and OR51B5/6 locus on chromosome 11p, due in part to the virtual absence of the Senegal and Indian Arab haplotypes. We also found evidence that selected SNPs in HBS1L-MYB region (HMIP) and BCL11A affect both other hematological indices and rates of hospitalization.ConclusionsThis study has confirmed the associations of SNPs in BCL11A and HBS1L-MYB and fetal haemoglobin in Cameroonian SCA patients; hematological indices and hospitalization rates were also associated with specific allelic variants.
Project description:Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity.
Project description:BackgroundFetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia.Methods and resultsSix BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%.ConclusionsCommon HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.
Project description: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.
Project description:Genetic studies have identified common variants within the HBS1L-MYB intergenic region on chromosome 6q associated with elevated fetal hemoglobin (HbF) levels and other clinically important human erythroid traits. The mechanism by which the non-coding sequence variants affect these traits is still not clear. Here we report that several of the variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We show that several of the variants reduce transcription factor binding, affecting long-range interactions with MYB, and MYB expression levels. We provide here a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a bona fide target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity.
Project description:We aimed to investigate the distribution of selected BCL11A and HMIP polymorphisms (SNP's), and to assess the correlation with HPFH in a cohort of sickle cell patients. A preliminary cross-sectional study was conducted in 102 patients. Group 1 was composed of patients with HPFH and Group 2 consisted of patients without HbF. We assessed 8 SNPs previously associated with HPFH in cohorts genetically close to the Congolese population. Observed frequencies were compared to expected frequencies. In the group 1, at rs7606173, the observed frequency for the genotype GG was significantly higher and the genotype GC was significantly lower than their respective expected frequencies. At rs9399137, the observed frequency of the genotype TT was significantly lower than expected. Conversely, the observed frequency of the genotype TC was significantly higher than expected. The observed frequency of the genotype TT at rs11886868 was significantly lower than the expected whereas the frequency of the genotype TC was significantly higher than observed. The lowest HbF level was recorded in patients with genotype CC at rs11886868. In this preliminary study, the results demonstrate that alleles of some of the 8 studied SNPs are not randomly distributed among patients with or without HPFH in this cohort.
Project description:Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10-44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (β = -1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.
Project description:The human ?-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre- and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or ?-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression.