Influence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the HBG2 XmnI polymorphism on Hb F levels

Abstract: The 1000 Genome project paved the way for sequencing diverse human populations. New genome projects are being established to sequence underrepresented populations helping in understanding human genetic diversity. The Kuwait Genome Project an initiative to sequence individual genomes from the three subgroups of Kuwaiti population namely, Saudi Arabian tribe; “tent-dwelling” Bedouin; and Persian, attributing their ancestry to different regions in Arabian Peninsula and to modern-day Iran (West Asia). These subgroups were in line with settlement history and are confirmed by genetic studies. In this work, we report whole genome sequence of a Kuwaiti native from Persian subgroup at >37X coverage. We document 3,573,824 SNPs, 404,090 insertions/deletions, and 11,138 structural variations. Out of the reported SNPs and indels, 85,939 are novel. We identify 295 ‘loss-of-function’ and 2,314 ’deleterious’ coding variants, some of which carry homozygous genotypes in the sequenced genome; the associated phenotypes include pharmacogenomic traits such as greater triglyceride lowering ability with fenofibrate treatment, and requirement of high warfarin dosage to elicit anticoagulation response. 6,328 non-coding SNPs associate with 811 phenotype traits: in congruence with medical history of the participant for Type 2 diabetes and β-Thalassemia, and of participant’s family for migraine, 72 (of 159 known) Type 2 diabetes, 3 (of 4) β-Thalassemia, and 76 (of 169) migraine variants are seen in the genome. Intergenome comparisons based on shared disease-causing variants, positions the sequenced genome between Asian and European genomes in congruence with geographical location of the region. On comparison, bead arrays perform better than sequencing platforms in correctly calling genotypes in low-coverage sequenced genome regions however in the event of novel SNP or indel near genotype calling position can lead to false calls using bead arrays. We report, for the first time, reference genome resource for the population of Persian ancestry. The resource provides a starting point for designing large-scale genetic studies in Peninsula including Kuwait, and Persian population. Such efforts on populations under-represented in global genome variation surveys help augment current knowledge on human genome diversity.

Abstract: The median age at diagnosis of patients with acute myeloid leukemia (AML) is 66 years, and most patients are > 60 years of age.[1] Due to comorbidities and adverse-risk disease, many older patients may not benefit from intensive chemotherapy.[2, 3] Hypomethylating agents such 5-azacytidine (AZA) and decitabine (DAC) have been used as “lower-intensity” therapy in older patients with AML and may be associated with outcomes similar to intensive therapy.[4, 5] In a post-hoc analysis of a randomized phase III trial of AZA vs. conventional care regimens (CCR) in patients with higher-risk myelodysplastic syndrome (MDS) and 20–30% blasts, AZA produced a response rate of 18% and was associated with an improvement in overall survival (OS) compared to CCR (24.5 vs. 16 months, p=0.001).[6] Subsequent reports have confirmed the activity of AZA in older patients with AML and in those unfit for intensive chemotherapy.[4, 7–9]

Abstract: Although the molecular basis of variability of hemoglobin (Hb) F has been extensively examined in β-thalassemia and sickle cell diseases, less study has been done on Hb E disorder. To address the variability of Hb F expression in Hb EE disease, we have examined multiple single nucleotide polymorphisms (SNPs) in the β-globin gene cluster, BCL11A and HBS1L-MYB genes and determined their associations with Hb F levels in this syndrome. Study was done on 141 adult Thai individuals with homozygous Hb E. Hematological parameters were recorded and Hb F measured using Hb-HPLC analyzer. It was found in 26 cases that co-inheritance of α-thalassemia could lead to significant lower production of Hb F. Association of Hb F expression with the Gγ-Xmn I polymorphism and other SNPs including rs2297339, rs2838513, rs4895441 and rs9399137 in HBS1L-MYB gene and rs4671393 and rs11886868 in BCL11A gene was therefore analyzed in the remaining 115 cases without α-thalassemia. It was found that 4 of these 7 SNPs including Gγ-XmnI polymorphism (rs7482144), HBS1L-MYB (rs4895441) and (rs9399137) and BCL11A (rs4671393) were significantly associated with higher proportions of subjects with high Hb F (Hb F ≥ 5%). The result demonstrated that multiple genetic modifying factors including T allele of Gγ-XmnI polymorphism (rs7482144), G allele of HBS1L-MYB (rs489441), C allele of HBS1L-MYB (rs9399137) and C allele of BCL11A (rs4671393) are associated with increased Hb F and in combination could explain approximately 80% of the variation of Hb F in Hb EE disease in Thai population. Other genetic factors regulating Hb F expression in this common genetic disorder remains to be elucidated.

Abstract: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are important biomarkers for disease development and progression. To gain insight into the genetic causes of variance in NLR and PLR in the general population, we conducted genome-wide association (GWA) analyses and estimated SNP heritability in a sample of 5901 related healthy Dutch individuals. GWA analyses identified a new genome-wide significant locus on the HBS1L-MYB intergenic region for PLR, which replicated in a sample of 2538 British twins. For platelet count, we replicated three known genome-wide significant loci in our cohort (at CCDC71L-PIK3CG, BAK1 and ARHGEF3). For neutrophil count, we replicated the PSMD3 locus. For the identified top SNPs, we found significant cis and trans expression quantitative trait loci effects for several loci involved in hematological and immunological pathways. Linkage Disequilibrium score (LD) regression analyses for PLR and NLR confirmed that both traits are heritable, with a polygenetic SNP heritability for PLR of 14.1%, and for NLR of 2.4%. Genetic correlations were present between ratios and the constituent counts, with the genetic correlation (r=0.45) of PLR with platelet count reaching statistical significance. In conclusion, we established that two important biomarkers have a significant heritable SNP component, and identified the first genome-wide locus for PLR.

Abstract: complications. JAK2V617F mutation primarily affects the proliferation of the red cell lineage but its impact on the phenotype of circulating RBCs is poorly documented. Our group showed increased adhesion of PV RBCs to endothelial cells mediated by erythroid Lu/BCAM and endothelial laminin a5 chain [4]. Recently, we showed that this abnormal adhesion stems from JAK2V617F-mediated Lu/BCAM activation through a Rap1/Akt signaling pathway [5]. Blocking this pathway by JAK2 specific inhibitors led to significant decrease of PV RBC adhesion [5]. Here, we extended our findings by a quantitative analysis to determine the relationship between the rate of RBC adhesion to laminin and the JAK2V617F allele burden. We first asked whether variation between patients in RBC adhesion was related to Lu/BCAM expression on the surface of PV RBCs. Indeed, Lu/BCAM erythroid expression is heterogeneous within the same individual, including a variable surface expression level and a proportion of Lu/BCAM-negative RBCs. We performed adhesion assays with blood samples from 17 JAK2V617F-positive PV patients, with no cytotoxic antiproliferative treatment, and determined the number of RBCs adhering to laminin at 3 dyn/cm. The adhesion values were variable, ranging from 14 to 1021 RBCs/mm (Supporting Information Table S1). The percentage of Lu/BCAM-positive RBCs as assessed by flow cytometry ranged from 38% to 90%, and the expression level, estimated by the mean fluorescence intensity (MFI), from 1249 to 3153 (Supporting Information Table S1). The number of adherent RBCs was plotted against the percentage of Lu/BCAM-positive RBCs or the MFI of each blood sample. We found no correlation between the number of adherent RBCs and the percentage of Lu/BCAM-positive RBCs (r 5 0.468 and P 5 0.058 at 3 dyn/cm, Fig. 1A), and between RBC adhesion and Lu/BCAM MFI values (r 5 0.345 and P 5 0.174 at 3 dyn/cm, Fig. 1B). These results indicated that Lu/ BCAM expression alone did not account for the variability of PV RBC adhesion, in accordance with the role of Lu/BCAM phosphorylation in the adhesion of PV RBCs to laminin [5]. Because Lu/BCAM phosphorylation is driven by a JAK2V617F-dependent pathway in PV RBCs [5], we asked whether RBC adhesion was influenced by JAK2V617F expression level. To date the most commonly used tool for evaluating the mutational load in one individual is by measuring the mutant allele burden in blood cell DNA. The JAK2V617F allele burden was determined for the 17 PV patients using an allele specific quantitative PCR (MutaquantVR , Qiagen) and ranged from 20 to 68% (Supporting Information Table S1). RBC adhesion values were plotted against the percentage of JAK2V617F. Statistical analysis showed a significant correlation between RBC adhesion and JAK2V617F percentage (r 5 0.571 and P 5 0.016, Fig. 1C) that was improved when the interval of Lu/ BCAM-positive RBCs was restricted to 65–90% (r 5 0.849 and P 5 0.0009, n 5 11 patients), indicating that for equivalent Lu/BCAM expression, higher JAK2V617F levels would drive higher RBC adhesion to laminin (Fig. 1D). As RBCs are enucleated, it is not possible to estimate the level of JAK2V617F using DNA or RNA based PCR assays. However, clonogenic assays showed that patients with higher JAK2V617F mutant allele burden in blood DNA carried more mutated erythroid progenitor cells [6]. Our study revealed RBC adhesion as a biological marker to estimate the V617F mutational load at the protein level in PV RBCs; although indirect, this marker has the potential of informing about the functional level of JAK2V617F in the circulation. This functional assay should be evaluated in a prospective study because it is a rapid and routinely manageable tool to estimate the mutant kinase activity in PV RBCs, which may help assessing the disease progression and the risk of evolution, in particular of thrombotic events in which abnormal adhesion of JAK2V617F-positive RBCs to endothelium could play a role. Furthermore, such a tool may be important to monitor the efficacy of JAK2 inhibitors in PV patients during the very early stages of their treatment by assessing the effect of these inhibitors on JAK2V617F activity in RBCs very shortly after their administration giving important information on their pharmacokinetics in the blood circulation. A European patent application (n8 EP13305131.8) was filed for this study, with the international extension n8 PCT/EP2014/052153.

Abstract: Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.

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.

Abstract: The beta-thalassaemia mutations in 702 unrelated carriers originating from seven different regions of the Indian subcontinent have been characterized using allele specific priming of the polymerase chain reaction (PCR). It was possible to identify the mutations in 688 (98%) of the individuals studied. Eleven different mutations were identified, of which five common ones accounted for 93.6%; namely the ones at IVS-1 position 5 (G-C), codons 8/9 (+G), IVS-1 position 1 (G-T), codons 41/42 (-CTTT) and the 619 bp deletion at the 3' end of the gene. The mutations at IVS-2 position 1 (G-A) and codon 30 (G-C), previously undescribed in Asian Indians, were found in two and six individuals respectively. Some regional variation in the distribution of beta-thalassaemia alleles was noted. These findings should prove useful for the development of a first trimester prenatal diagnosis programme based on direct detection of mutations.

Abstract: 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'.

Abstract: As the defective genes for more and more genetic disorders become unravelled, it is clear that patients with apparently identical genotypes can have many different clinical conditions even in simple monogenic disorders. Beta thalassemia occurs when there is a deficiency in the synthesis of beta globin chains. The clinical manifestations of beta thalassemia are extremely diverse, spanning a broad spectrum from severe anemia and transfusion-dependency to the asymptomatic state of thalassemia trait. The remarkable phenotypic diversity of the beta thalassemias is prototypical of how a wide spectrum of disease severity can be generated in single gene disorders. The most reliable and predictive factor of disease phenotype is the nature of the mutation at the beta globin locus itself. However, relating phenotype to genotype is complicated by the complex interaction of the environment and other genetic factors at the secondary and tertiary levels, some implicated from family studies, and others, as yet unidentified. This article reviews the clinical and hematologic diversity encountered in beta thalassemia with an overview of the modifier genes that moderate their disease expression.