Genetic variants at HbF-modifier loci moderate anemia and leukocytosis in sickle cell disease in Tanzania.
ABSTRACT: Fetal hemoglobin (HbF) is a recognized modulator of sickle cell disease (SCD) severity. HbF levels are strongly influenced by genetic variants at three major genetic loci, Xmn1-HBG2, HMIP-2, and BCL11A, but the effect of these loci on the hematological phenotype in SCD, has so far not been investigated. In a cohort of individuals with SCD in Tanzania (HbSS and HbS/β° thalassemia, n = 726, aged 5 or older), HbF levels were positively correlated with hemoglobin, red blood cell (RBC) indices, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH), and negatively with white blood cell (WBC) and platelet counts (all P < 0.0001). We subsequently assessed the contribution of the three HbF modifier loci and detected diverse effects, including a reduction in anemia, leukocytosis, and thrombocytosis associated with certain HbF-promoting alleles. The presence of the 'T' allele at Xmn1-HBG2 led to a significant increase in hemoglobin (P = 9.8 × 10(-3) ) but no changes in cellular hemoglobin content. Xmn1-HBG2 'T' also has a weak effect decreasing WBC (P = 0.06) and platelet (P = 0.06) counts. The BCL11A variant (rs11886868-'C') increases hemoglobin (P = 2 × 10(-3) ) and one of the HBS1L-MYB variants decreases WBC values selectively (P = 2.3 × 10(-4) ). The distinct pattern of effects of each variant suggests that both, disease alleviation through increased HbF production, and 'pleiotropic' effects on blood cells, are involved, affecting a variety of pathways.
Project description:High levels of fetal hemoglobin (HbF) reduce sickle cell anemia (SCA) morbidity and mortality. HbF levels vary considerably and there is a strong genetic component that influences HbF production. Genetic polymorphisms at three quantitative trait loci (QTL): Xmn1-HBG2, HMIP-2 and BCL11A, have been shown to influence HbF levels and disease severity in SCA. Hydroxyurea (HU) is a drug that increases HbF. We investigated the influence of single nucleotide polymorphisms (SNPs) at the Xmn1-HBG2 (rs7482144); BCL11A (rs1427407, rs4671393 and rs11886868); and HMIP-2 (rs9399137 and rs9402686) loci on baseline and HU-induced HbF levels in 111 HbSS patients. We found that both BCL11A and HMIP-2 were associated with increased endogenous levels of HbF. Interestingly, we also found that BCL11A was associated with higher induction of HbF with HU. This effect was independent of the effect of BCL11A on baseline HbF levels. Additional studies will be needed to validate these findings and explain the ample inter-individual variations in HbF levels at baseline and HU-induced in patients with SCA.
Project description:Fetal hemoglobin (HbF) is a strong modifier of sickle cell disease (SCD) severity and is associated with 3 common genetic loci. Quantifying the genetic effects of the 3 loci would specifically address the benefits of HbF increases in patients. Here, we have applied statistical methods using the most representative variants: rs1427407 and rs6545816 in BCL11A, rs66650371 (3-bp deletion) and rs9376090 in HMIP-2A, rs9494142 and rs9494145 in HMIP-2B, and rs7482144 (Xmn1-HBG2 in the ?-globin locus) to create g(HbF), a genetic quantitative variable for HbF in SCD. Only patients aged ?5 years with complete genotype and HbF data were studied. Five hundred eighty-one patients with hemoglobin SS (HbSS) or HbS?0 thalassemia formed the "discovery" cohort. Multiple linear regression modeling rationalized the 7 variants down to 4 markers (rs6545816, rs1427407, rs66650371, and rs7482144) each independently contributing HbF-boosting alleles, together accounting for 21.8% of HbF variability (r2) in the HbSS or HbS?0 patients. The model was replicated with consistent r2 in 2 different cohorts: 27.5% in HbSC patients (N = 186) and 23% in 994 Tanzanian HbSS patients. g(HbF), our 4-variant model, provides a robust approach to account for the genetic component of HbF in SCD and is of potential utility in sickle genetic and clinical studies.
Project description: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:BACKGROUND:Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation. METHODS:We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (?7.7% for high HbF) and (?2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1. RESULTS:Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. CONCLUSIONS:This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.
Project description:Increased levels of fetal hemoglobin (HbF, alpha(2)gamma(2)) are of no consequence in healthy adults, but confer major clinical benefits in patients with sickle cell anemia (SCA) and beta thalassemia, diseases that represent major public health problems. Inter-individual HbF variation is largely genetically controlled, with one extreme caused by mutations involving the beta globin gene (HBB) complex, historically referred to as pancellular hereditary persistence of fetal hemoglobin (HPFH). These Mendelian forms of HPFH are rare and do not explain the common form of heterocellular HPFH which represents the upper tail of normal HbF variation, and is clearly inherited as a quantitative genetic trait. Genetic studies have identified three major quantitative trait loci (QTLs) (Xmn1-HBG2, HBS1L-MYB intergenic region on chromosome 6q23, and BCL11A on chromosome 2p16) that account for 20-50% of the common variation in HbF levels in patients with SCA and beta thalassemia, and in healthy adults. Two of the major QTLs include oncogenes, emphasizing the importance of cell proliferation and differentiation as an important contribution to the HbF phenotype. The review traces the story of HbF quantitative genetics that uncannily mirrors the changing focus in genetic methodology, from candidate genes through positional cloning, to genome-wide association, that have expedited the dissection of the genetic architecture underlying HbF variability. These genetic results have already provided remarkable insights into molecular mechanisms that underlie the hemoglobin 'switch'.
Project description:Patients with inherited bone marrow failure syndromes (IBMFS) have 'stress erythropoiesis', with anaemia, macrocytosis, increased fetal haemoglobin (Hb F) and high erythropoietin levels. In haemoglobinopathies, Hb F levels are regulated by 3 quantitative trait loci, HBS1L-MYB, BCL11A and Xmn1-HBG2. In our study of 97 patients with an IBMFS, increased Hb F was associated with young age, male gender, anaemia, high erythropoietin levels, and alternative alleles in Xmn1-HBG2 [adjusted P = 0·04 for the total group, driven by Fanconi anaemia (P = 0·02) and dyskeratosis congenita (P = 0·09)]. Thus Hb F is regulated in IBMFS by Xmn1-HBG2, as it is in the haemoglobinopathies.
Project description:Fetal hemoglobin (HbF) is a major modifier of disease severity in sickle cell anemia (SCA). Three major HbF quantitative trait loci (QTL) are known: the Xmn I site upstream of (G)?- globin gene (HBG2) on chromosome 11p15, BCL11A on chromosome 2p16, and HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23. However, the roles of these QTLs in patients with SCA with uncharacteristically high HbF are not known. We studied 20 African American patients with SCA with markedly elevated HbF (mean 17.2%). They had significantly higher minor allele frequencies (MAF) in two HbF QTLs, BCL11A, and HMIP, compared with those with low HbF. A 3-bp (TAC) deletion in complete linkage disequilibrium (LD) with the minor allele of rs9399137 in HMIP was also present significantly more often in these patients. To further explore other genetic loci that might be responsible for this high HbF, we sequenced a 14.1 kb DNA fragment between the (A)?-(HBG1) and ?-globin genes (HBD). Thirty-eight SNPs were found. Four SNPs had significantly higher major allele frequencies in the unusually high HbF group. In silico analyses of these four polymorphisms predicted alteration in transcription factor binding sites in 3.
Project description:Persistence of human fetal hemoglobin (HbF, ?(2)?(2)) in adults lessens the severity of sickle cell disease (SCD) and the ?-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of ?-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.
Project description:Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress ?-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ?-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2r?-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.
Project description:Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes ?-globin), mainly sickle cell disease (SCD) and ?-thalassemia, become symptomatic postnatally as fetal ?-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult ?-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or ?2?2) to adult (referred to as HbA or ?2?2) form. These disorders are alleviated when postnatal expression of fetal ?-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate ?-globin-to-?-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with ?-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of ?-hemoglobinopathies.