Genetic association studies in β-hemoglobinopathies

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 354-361 ◽  
Author(s):  
Swee Lay Thein
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Clinical Severity ◽  
Genetic Modifiers ◽  
Genetic Studies ◽  
Globin Gene Regulation ◽  
Innate Ability

Abstract Characterization of the molecular basis of the β-thalassemias and sickle cell disease (SCD) clearly showed that individuals with the same β-globin genotypes can have extremely diverse clinical severity. Two key modifiers, an innate ability to produce fetal hemoglobin and coinheritance of α-thalassemia, both derived from family and population studies, affect the pathophysiology of both disorders at the primary level. In the past 2 decades, scientific research had applied genetic approaches to identify additional genetic modifiers. The review summarizes recent genetic studies and key genetic modifiers identified and traces the story of fetal hemoglobin genetics, which has led to an emerging network of globin gene regulation. The discoveries have provided insights on new targets for therapeutic intervention and raise possibilities of developing fetal hemoglobin predictive diagnostics for predicting disease severity in the newborn and for integration into prenatal diagnosis to better inform genetic counseling.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1839-1842 ◽  
Author(s):  
Vijay G. Sankaran ◽  
Tobias F. Menne ◽  
Jian Xu ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
...  
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Sequence Variants ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Direct Role ◽  
Globin Gene Regulation

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-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 β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

scholarly journals Debunking the "junk": Unraveling the role of lncRNA–miRNA–mRNA networks in fetal hemoglobin regulation

2021 ◽  
Author(s):  
Motiur Rahaman ◽  
Chiranjib Bhowmick ◽  
Jaikrishna Komanapalli ◽  
Mandrita Mukherjee ◽  
Prasanna Kumar Byram ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
In Silico Analysis ◽  
Clinical Severity ◽  
Differentially Expressed ◽  
Supervised Machine Learning ◽  
Pharmacological Intervention ◽  
Differentially Expressed Mirnas ◽  
Hbf Induction ◽  
Globin Gene Regulation

Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of β-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of γ-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

FOXO3 Variants Are Associated With Lower Fetal Hemoglobin Levels In Children With Sickle Cell Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 778-778 ◽  
Author(s):  
Vivien A Sheehan ◽  
Jacy R Crosby ◽  
Aniko Sabo ◽  
Thad A Howard ◽  
Donna M. Muzny ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Association Studies ◽  
Single Gene ◽  
Phosphorylation Site ◽  
Fetal Hemoglobin ◽  
Clinical Severity ◽  
Genome Wide Association Studies ◽  
Genetic Modifiers ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia (SCA) exhibit wide variability in their degree of clinical severity. One of the most powerful and reproducible predictors of disease severity is the level of endogenous fetal hemoglobin (HbF). Several genetic modifiers of HbF levels have previously been identified, by association with beta globin haplotypes, or by genome wide association studies. These methods are only able to detect common variants, with a minor allele frequency (MAF) greater than 5%. We have applied whole exome sequencing (WES) to find new genetic variants associated with baseline HbF in SCA, using 171 pediatric SCA genomes from participants in two clinical trials, HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980). WES allows identification of both common and rare exonic variants, and use of burden analysis testing. In order to capture the association between the phenotype variants with a MAF below 1%, burden tests maximize power by grouping low frequency variants together by gene. This allows us to transition from a single variant theory, in which a variant is associated with a phenotype, to a gene-based theory, in which a collection of rare variants within a single gene are associated with the phenotype. Burden analysis (T1), found seven unique non-synonymous variations in a Forkhead box O transcription factor, FOXO3, to be significantly associated with lower HbF (p=5.6x10-4, b-value ln HbF -0.66). All variants produced the same effect, a lowering of HbF. HbF values were normalized using natural log transformation to permit analysis, and adjusted for age, BCL11A, and XmnI variant status. The box-plot below shows the overall effect of any of the non-synonymous exonic variants in FOXO3 on baseline HbF compared to wild-type. Each individual was heterozygous for a variant. FOXO3 is involved in multiple cellular processes, including cell cycle arrest, removal of reactive oxygen species, and regulation of erythroid differentiation. Erythroid maturation is altered by changes in post-translational maturation (PTM) such as removal of a phosphorylation site, as the in Ser553Phe variant identified in our analysis (Bakker, et al, JCB 2004). Changes in erythroid maturation kinetics may affect the amount of HbF produced. Variations in FOXO3 may represent another contributor to the heritability of HbF in patients with SCA. Further functional genomics analysis of FOXO3 may add to our understanding of gamma globin induction.Table 1Description of FOXO3 variants found to be associated with lower HbF in a cohort of pediatric sickle cell patients by WES and burden testing (T1).Chr 6LocationAmino Acid ChangeAltered PTMPolyPhen2 prediction%MAF108882607Asp66Asnnone knownPossibly damaging0.3108882830Ala140Valnone knownBenign0.6108984883Asp283Asnnone knownDamaging0.3108985057Ala121Thrnone knownBenign0.3108985280Pro415Leunone knownDamaging0.6108985679Arg548HismethylationDamaging0.3108985694Ser553PhephosphorylationBenign0.3Figure 1Boxplot demonstrating effect of any FOXO3 variants on endogenous %HbF compared to wild-type individuals.Figure 1. Boxplot demonstrating effect of any FOXO3 variants on endogenous %HbF compared to wild-type individuals. Disclosures: Off Label Use: Hydroxyurea is not FDA approved for use in pediatric sickle cell patients.

scholarly journals Random Forests for Genetic Association Studies

2011 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin A Goldstein ◽  
Eric C Polley ◽  
Farren B. S. Briggs
Keyword(s):  
Machine Learning ◽  
Genetic Association ◽  
Random Forests ◽  
Learning Algorithm ◽  
Association Studies ◽  
Genetic Association Studies ◽  
Machine Learning Algorithms ◽  
Computationally Efficient ◽  
Genetic Studies ◽  
Variable Importance Measures

The Random Forests (RF) algorithm has become a commonly used machine learning algorithm for genetic association studies. It is well suited for genetic applications since it is both computationally efficient and models genetic causal mechanisms well. With its growing ubiquity, there has been inconsistent and less than optimal use of RF in the literature. The purpose of this review is to breakdown the theoretical and statistical basis of RF so that practitioners are able to apply it in their work. An emphasis is placed on showing how the various components contribute to bias and variance, as well as discussing variable importance measures. Applications specific to genetic studies are highlighted. To provide context, RF is compared to other commonly used machine learning algorithms.

Searching for Disease Modifiers Genes in Thalassemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3642-3642
Author(s):  
Suthat Fucharoen ◽  
Orapan Sripichai ◽  
Pranee Winichagoon ◽  
Kenneth Abel ◽  
Andreas Braun
Keyword(s):  
Disease Severity ◽  
Dna Analysis ◽  
Genome Wide Association Study ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Nucleotide Polymorphisms ◽  
Genetic Modifiers ◽  
Chromosome 11 ◽  
Genotype Frequencies ◽  
A Genome

Abstract β0-thalassemia/HbE disease is one of the most common thalassemias in Southeast Asia. Patients with compound heterozygote for HbE and β0 mutant alleles display remarkable variability in clinical expression, ranging from nearly asymptomatic to severe, transfusion-dependent disease. It is believed that additional genetic factors modifying disease severity may account for this variability. A universal platform technology (MassARRAY) based on mass spectrometry for analyzing nucleic acids at a high level of precision and accuracy has been developed. To identify genetic modifiers influencing severity among 1060 β0-thalassemia/HbE patients, we are utilizing this technology to conduct a genome-wide association study involving approximately 110,000 gene-based single nucleotide polymorphisms (SNPs). This assay panel corresponds to SNPs with a median spacing of 10.4 kilobases, in approximately 99% of all known and predicted human genes. DNAs from approximately 200 regionally matched patients representing the extremes of mild and severe cases were included in each DNA pool. Allele frequencies for all SNPs were estimated in both pools, and those showing suggestive significant differences (p values <0.02) were selected for verification by repeated pooled DNA analysis. Approximately one-fourth of these showed reproducible allelic differences at p<0.05, and more than 600 were selected for genotyping the individual patient DNAs in order to determine precise allele and genotype frequencies. A number of SNPs showed evidence for association with disease severity, including several in reported quantitative trait loci (QTLs) associated with fetal hemoglobin HbF levels. However the most strongly associated SNPs were within a region on chromosome 11 distinct from the beta globin gene cluster, within which most analysis to date has focused.

Control of Hemoglobin Switching by BCL11A.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5-5
Author(s):  
Jian Xu ◽  
Vijay G. Sankaran ◽  
Yuko Fujiwara ◽  
Stuart H. Orkin
Keyword(s):  
Gene Silencing ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Association Studies ◽  
Globin Gene ◽  
Clinical Severity ◽  
Genome Wide Association Studies ◽  
Erythroid Cells ◽  
B Locus

Abstract Abstract 5 All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals α-Globin as a molecular target in the treatment of β-thalassemia

Blood ◽  
2015 ◽  
Vol 125 (24) ◽  
pp. 3694-3701 ◽  
Author(s):  
Sachith Mettananda ◽  
Richard J. Gibbons ◽  
Douglas R. Higgs
Keyword(s):  
Globin Gene ◽  
Molecular Genetic ◽  
Allogeneic Bone Marrow Transplantation ◽  
Fetal Hemoglobin ◽  
Developed Countries ◽  
Allogeneic Bone ◽  
Genetic Studies ◽  
Hemoglobin E ◽  
Premature Deaths ◽  
Globin Gene Expression

Abstract The thalassemias, together with sickle cell anemia and its variants, are the world’s most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia.

scholarly journals Dissecting the role of genetic modifiers of hemoglobinopathies: A futuristic approach towards precision medicine

2021 ◽  
Author(s):  
Priya Hariharan ◽  
Manju Gorivale ◽  
Pratibha Sawant ◽  
Pallavi Mehta ◽  
Anita Nadkarni
Keyword(s):  
Disease Severity ◽  
Phenotypic Variability ◽  
Globin Gene ◽  
Gene Promoter ◽  
Clinical Severity ◽  
Genetic Modifiers ◽  
Promoter Polymorphisms ◽  
Cumulative Impact ◽  
Remarkable Result

Abstract Introduction: Hemoglobinopathies though a monogenic disorder, show phenotypic variability. Hence, understanding the genetics underlying the heritable sub-phenotypes of hemoglobinopathies, specific to each population, would be prognostically useful and could inform personalized therapeutics. This study aimed to evaluate the role of genetic modifiers leading to higher HbF production with cumulative impact of the modifiers on disease severity. Materials and methods:200 patients [100 β-thalassemia homozygotes,100 Sickle Cell Anemia], and 50 healthy controls were recruited. Primary screening followed with molecular analysis for confirming the β-hemoglobinopathy was performed. Co-existing α-thalassemia and the polymorphisms located in 3 genetic loci linked to HbF regulation were screened.Results: The most remarkable result was the association of SNPs with clinically relevant phenotypic groups. The γ-globin gene promoter polymorphisms [-158 C→T,+25 G→A],BCL11A rs1427407 G→T,-3 bp HBS1L-MYB rs66650371 and rs9399137 T→C polymorphisms were correlated with higher HbF, in group that has lower disease severity score (P<0.00001), milder clinical presentation, and a significant delay in the age of the first transfusion.Conclusion:Our study emphasizes the complex genetic interactions underlying the disease phenotype that may be a prognostic marker for predicting the clinical severity and assist in disease management.

scholarly journals Genotype - Phenotype Correlation Among Haemoglobin E β-thalassaemia Patients

2021 ◽  
Author(s):  
Farin Masra ◽  
Siti Razak ◽  
Nor Murad ◽  
Doris Chong ◽  
C-Khai Loh ◽  
...  
Keyword(s):  
Haemoglobin Level ◽  
Globin Gene ◽  
Cross Sectional Study ◽  
Clinical Severity ◽  
Genetic Modifiers ◽  
Clinical Predictors ◽  
Cross Sectional ◽  
Gene Deletions ◽  
Severity Scoring ◽  
Severe Group

Abstract Background and objectives: Haemoglobin E β-thalassaemia has a variable clinical presentation. This study describes the clinical spectrum of these patients in two thalassaemia centers in Malaysia in addition to determining the prevalence of selected primary and secondary genetic modifiers which may influence its phenotype. Methods: A total of 99 patients were recruited in this cross-sectional study. Clinical parameters and severity scoring were determined. Molecular analysis was performed: Sanger sequencing and MLPA for β globin mutations; multiplex PCR for α-globin gene deletions and RFLP-PCR for XmnI polymorphism.Results: Patients with mild HbE β-thalassaemia were diagnosed at a later age as compared to the severe group (mean 3.14 and 1.6 years, p = 0.03). Haemoglobin level at diagnosis was higher for the mild group as compared to severe group (7.9 g/dL ± 1.97 and 6.0 g/dL ± 1.00, p = 0.02). The commonest β mutation in Malays were IVS1-5 (51.69%) and CD41/42 (20.2%) whereas in the Chinese, IVS2-654(44.4%) and CD41/42(33.4%). Single α-gene deletion (-α3.7/αα) was found in 4% of patients and none were homozygous for XmnI (+/+) polymorphism. Conclusion: Age at presentation and haemoglobin level at initial diagnosis is useful as clinical predictors of disease severity. The majority of our patients had β° gene mutation i.e. IVS1-5 and CD41/42, which accounted for the moderate to severe phenotype based on clinical severity scoring.

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