Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements, may require several different labor-intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here we describe a next generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for ???? thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the ?-locus control region. Identification of reads spanning the breakpoints provided to-the-base resolution of the rearrangement, subsequently confirmed by gap-PCR and Sanger sequence analysis. The same rearrangement, termed Inv-Del English V ???? thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders. This article is protected by copyright. All rights reserved.
Renal disease is one of the most frequent and severe complications experienced by patients with sickle cell disease; its prevalence is likely to increase as the patient population ages. We recommend regular monitoring for early signs of renal involvement and a low threshold for the use of hydroxyurea as preventative measures for end-stage renal disease. Once renal complications are detected, a careful assessment of the patient is required to rule out other causes of renal disease. Proteinuria and hypertension should be managed aggressively and the patient referred to a specialist nephrology center when progressive decline in renal function is noted. For the few patients who develop advanced chronic kidney disease, timely planning for dialysis and transplantation can significantly improve outcome, and we recommend an exchange blood transfusion policy for all patients on the transplant waiting list and for those with a functioning graft. Alongside the invasive treatment regimes, it is important to remember that renal failure in conjunction with sickle cell disease does carry a significant burden of morbidity and that focusing on symptom control has to be central to good patient care.
To test the hypothesis that vascular abnormalities on high-resolution CT (HRCT) would be associated with echocardiographic changes and lung function abnormalities in patients with sickle cell disease (SCD) and the decline in lung function seen in SCD patients.
HMIP-2 is a human quantitative trait locus affecting peripheral numbers, size and hemoglobin composition of red blood cells, with a marked effect on the persistence of the fetal form of hemoglobin, HbF, in adults. The locus consists of multiple common variants in an enhancer region for MYB (chr 6q23.3), which encodes the hematopoietic transcription factor cMYB. Studying a European population cohort and four African-descended groups of patients with sickle cell anemia, we found that all share a set of two spatially separate HbF-promoting alleles at HMIP-2, termed "A" and "B." These typically occurred together ("A-B") on European chromosomes, but existed on separate homologous chromosomes in Africans. Using haplotype signatures for "A" and "B," we interrogated public population datasets. Haplotypes carrying only "A" or "B" were typical for populations in Sub-Saharan Africa. The "A-B" combination was frequent in European, Asian, and Amerindian populations. Both alleles were infrequent in tropical regions, possibly undergoing negative selection by geographical factors, as has been reported for malaria with other hematological traits. We propose that the ascertainment of worldwide distribution patterns for common, HbF-promoting alleles can aid their further genetic characterization, including the investigation of gene-environment interaction during human migration and adaptation.
Sickle cell disease (SCD) has evolved into a debilitating disorder with emerging end-organ damage. One of the organs affected is the liver, causing "sickle hepatopathy," an umbrella term for a variety of acute and chronic pathologies. Prevalence of liver dysfunction in SCD is unknown, with estimates of 10%. Dominant etiologies include gallstones, hepatic sequestration, viral hepatitis, and sickle cell intrahepatic cholestasis (SCIC). In addition, causes of liver disease outside SCD must be identified and managed. SCIC is an uncommon, severe subtype, with outcome of its acute form having vastly improved with exchange blood transfusion (EBT). In its chronic form, there is limited evidence for EBT programs as a therapeutic option. Liver transplantation may have a role in a subset of patients with minimal SCD-related other organ damage. In the transplantation setting, EBT is important to maintain a low hemoglobin S fraction peri- and posttransplantation. Liver dysfunction in SCD is likely to escalate as life span increases and patients incur incremental transfusional iron overload. Future work must concentrate on not only investigating the underlying pathogenesis, but also identifying in whom and when to intervene with the 2 treatment modalities available: EBT and liver transplantation.
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 ?-thalassemia disease severity.
Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.
Characterization of the molecular basis of the ?-thalassemias and sickle cell disease (SCD) clearly showed that individuals with the same ?-globin genotypes can have extremely diverse clinical severity. Two key modifiers, an innate ability to produce fetal hemoglobin and coinheritance of ?-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.
Abstract Mutations within the promoters of either of the ?-globin genes [(G)? (HBG1) and (A)? (HBG2)] lead to variably increased levels of fetal hemoglobin (Hb) (Hb F, ?2?2) in the syndrome of hereditary persistence of fetal Hb (HPFH). Carriers of such mutations are clinically asymptomatic and the mutations are usually detected as part of routine screening or family studies. We describe two new nondeletional HPFH mutations, both C>T substitutions at position c.-250, one in the HBG1 and the other in the HBG2 globin gene promoters.
The ?-thalassemias are characterized by a quantitative deficiency of ?-globin chains underlaid by a striking heterogeneity of molecular defects. Although most of the molecular lesions involve the structural ? gene directly, some down-regulate the gene through distal cis effects, and rare trans-acting mutations have also been identified. Most ?-thalassemias are inherited in a Mendelian recessive fashion but there is a subgroup of ?-thalassemia alleles that behave as dominant negatives. Unraveling the molecular basis of ?-thalassemia has provided a paradigm for understanding of much of human genetics.
Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome-wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10(-07) ). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)-33, HS-40 and HS-48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r(2) = 1) and in strong LD with rs7197554 (r(2) = 0·75) and rs13336641 (r(2) = 0·77); the latter is located between HS-33 and HS-40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the -?(3·7) thalassaemia gene deletion. When adjusting for HbF and ? thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion? thalassaemia (P = 0·02463). Perhaps by independently down-regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.
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.
The red blood cell related traits are highly heritable but their genetics are poorly defined. Only 5-10% of the total observed variance is explained by the genetic loci found to date, suggesting that additional loci should be searched using approaches alternative to large meta analysis. GWAS (Genome Wide Association Study) for red blood cell traits in a founder population cohort from Northern Italy identified a new locus for mean corpuscular hemoglobin concentration (MCHC) in the TAF3 gene. The association was replicated in two cohorts (rs1887582, P?=?4.25E-09). TAF3 encodes a transcription cofactor that participates in core promoter recognition complex, and is involved in zebrafish and mouse erythropoiesis. We show here that TAF3 is required for transcription of the SPTA1 gene, encoding alpha spectrin, one of the proteins that link the plasma membrane to the actin cytoskeleton. Mutations in SPTA1 are responsible for hereditary spherocytosis, a monogenic disorder of MCHC, as well as for the normal MCHC level. Based on our results, we propose that TAF3 is required for normal erythropoiesis in human and that it might have a role in controlling the ratio between hemoglobin (Hb) and cell volume and in the dynamics of RBC maturation in healthy individuals. Finally, TAF3 represents a potential candidate or a modifier gene for disorders of red cell membrane.
Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.
Studies have questioned whether renal dysfunction in sickle cell disease is linked to hemolysis-associated vasculopathy. We have investigated renal function and markers of hemolysis in a cohort of 424 adult African-British patients with sickle cell disease. While significant associations were found in HbSS and HbS?(0) (sickle cell anemia) patients with and without controlling for covariates between hemolytic markers and albuminuria, the associations were not significant in patients with HbSC. Estimated glomerular filtration rate, a marker of renal function, correlated significantly with reticulocyte count and bilirubin. Alpha thalassemia, present in 34% of the sickle cell anaemia patients, had a protective effect against albuminuria in this group. Altogether, the incidence of hyperfiltration was 71% and microalbuminuria 37%, making nephropathy a common complication of sickle cell anemia.
Sickle cell disease is one of the best characterized human monogenic disorders. Complex genotype/phenotype correlations clearly demonstrate the interaction of multiple genetic and environmental factors. In the last 20 years, scientific research has applied genetic approaches to dissect some of these modifiers. This review highlights the more recent genetic association studies that have been applied to unravel the genetic modifiers of sickle cell disease including Hb F genetics, and the key genetic variants identified. Illumination of such modifying factors may guide future therapeutic interventions and improve prediction of disease severity, with implications for genetic counseling, prenatal diagnosis and implementation of high risk therapy.
A study published in 1981 examined the causes of hospital admission for a cohort of children with sickle cell disease (SCD). Since that time, the incidence and prevalence of SCD has increased markedly in the UK, and there have been many changes in the management of this disease. We undertook a study examining the causes of hospital admission of children with SCD to the same hospital as the previous study, over the 2-year period from 2008 to 2009. We found that the numbers of children being cared for by our hospital had dramatically increased over the last 50 years, but rates of hospital admission had significantly fallen (41 hospital admissions per 100 patient-years, compared with 111.3 admissions per 100 patient-years in the original study). This fall in admission rates is accounted for by 2 major components: acute painful episodes (15.7 admissions per 100 patient-years compared with 39.3 in the previous study) and admission for elective blood transfusion (0.2 admissions per 100 patient-years compared with 26.7 in the previous study). It is interesting to note that, 541 elective transfusions were carried out during the study period, but in a day-care setting rather than requiring overnight admission. This study illustrates the changes in management of SCD over the past 30 years, and reflects the overall trend common to most hospital specialties of increasing community and ambulatory care.
Professor Titus H.J. Huisman is best known for his work on hemoglobin (Hb) variants. To date, more than 1,000 Hb variants have been discovered and characterized, of which about one-third were discovered in Titus Huismans laboratory at the Medical College of Georgia, Augusta, GA, USA. A registry of these Hb variants and other information, a legacy from Professor Huisman, is now available online, at HbVar database (hhtp://globin.bx.psu.edu/hbvar). During the last century, major developments in Hb research have been made using physical, chemical, physiological and genetic methods. This review highlights the milestones and key developments in Hb research most relevant to hematologists, and that have impacted our understanding and management of the thalassemias and sickle cell disease.
In patients who have inherited both the sickle cell gene and the ?-thalassemia (?-thal) gene, the nature of the ?-thal mutation will impact on the disease phenotype. The ?-thal mutation caused by the 1393 bp deletion has previously been described as having a mild clinical phenotype when inherited with the sickle gene. We describe three members of a family with this deletion who present with a more severe phenotype. The severity cannot be explained by their Hb F levels, or the XmnI-HBG2 polymorphism. This deletion cannot be presumed to be associated with a mild disease phenotype and we recommend that patients with Hb S/?(0)-thal are screened for this deletion.
It is widely assumed that, at matched transfusional iron-loading rates, responses to chelation therapy are similar, irrespective of the underlying condition. However, data are limited for rare transfusion-dependent anaemias, and it remains to be elucidated if response differs, depending on whether the anaemia has a primary haemolytic or production mechanism.
White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.
We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.
In patients with cyanosis from congenital heart disease, erythropoiesis is governed by many factors that can alter the expected relation between the oxygen saturation (O(2sat)) and hemoglobin concentration. We sought to define the relation between the O(2sat) and hemoglobin in such patients and to predict an ideal hemoglobin concentration for a given O(2sat). Adults with congenital heart defects and cyanosis were studied prospectively using blood tests and exercise testing. Nonoptimal hemoglobin was defined as any evidence of inadequate erythropoiesis (i.e., iron, folate, or vitamin B(12) deficiency, increased erythropoietin, reticulocytosis, or a right-shifted oxygen-hemoglobin curve). For patients without these factors, a linear regression equation of hemoglobin versus O(2sat) was used to predict the optimal hemoglobin for all patients. Of the 65 patients studied, 21 met all the prestudy criteria for an optimal hemoglobin. For all patients, no correlation was found between O(2sat) and hemoglobin (r = -0.22). However, a strong linear correlation was found for those meeting the criteria for optimal hemoglobin (r = -0.865, p <0.001). The optimal hemoglobin regression equation was as follows: predicted hemoglobin = 57.5 - (0.444 × O(2sat)). A negative correlation was found between the hemoglobin difference (predicted minus measured) and exercise duration on cardiopulmonary exercise testing (r = -0.396, p = 0.005) and the 6-minute walk distance (r = -0.468, p <0.001). In conclusion, a strong relation between O(2sat) and hemoglobin concentration can be shown in stable cyanotic patients and used to predict an optimal hemoglobin. This relation might be useful in defining functional anemia in this group.
Transfusion of red blood cells is a major therapeutic option in sickle cell disease (SCD). There is strong evidence for its efficacy, particularly in primary and secondary stroke prevention in children, however, its use in other areas remains controversial. This study assessed the patterns of transfusion in the adult cohort attending Kings College Hospital over a 10-year period, from 2000 to 2009. Total blood usage has increased significantly (P = 0·006) during this time, with 78% of the blood received by only 6% of the patients. The increase is explained by increased automated red cell exchange and increased usage for planned and acute transfusions for sickle-related complications.
Fetal hemoglobin (HbF, ?(2)?(2)) is a major contributor to the remarkable phenotypic heterogeneity of sickle cell anemia (SCA). Genetic variation at 3 principal loci (HBB cluster on chromosome 11p, HBS1L-MYB region on chromosome 6q, and BCL11A on chromosome 2p) have been shown to influence HbF levels and disease severity in ?-thalassemia and SCA. Previous studies in SCA, however, have been restricted to populations from the African diaspora, which include multiple genealogies. We have investigated the influence of these 3 loci on HbF levels in sickle cell patients from Tanzania and in a small group of African British sickle patients. All 3 loci have a significant impact on the trait in both patient groups. The results suggest the presence of HBS1L-MYB variants affecting HbF in patients who are not tracked well by European-derived markers, such as rs9399137. Additional loci may be identified through independent genome-wide association studies in African populations.
Although most common in tropical regions, population migration has meant that sickle cell disease is now one of the most prevalent genetic diseases worldwide. The issues and challenges faced by physicians and patients have been discussed by an international group of experts representing 4 key regions: the USA, Europe, Latin America, and the Middle East/Africa. Conclusive evidence to support the use of transfusion therapy for the prevention of stroke has resulted in key changes to patient management in all regions, and increasing numbers of patients are benefiting from this management approach. However, it is apparent that transfusion therapy is still under-utilized, largely due to concerns over iron overload, alloimmunization, limited blood supplies, and, sometimes, due to parental refusal. Once transfused, assessment and management of body iron levels can be poor, particularly in patients who are intermittently transfused. Compliance with chelation therapy regimens is a significant challenge, but new therapeutic options are likely to overcome some of the current barriers. Key requirements in all regions were considered to be the following: to provide greater physician, patient, and family education; to ensure effective transition from pediatric to adult care; and to establish national guidelines in order to ensure best practice is consistently applied.
Sickle cell anemia is caused by a single type of mutation, a homozygous A?T substitution in the ß globin gene. Clinical severity is diverse, partially due to additional, disease-modifying genetic factors. We are studying one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome 6q23.3). Working with a genetically admixed patient population, we have encountered the necessity to generate haplotype signatures of genetic markers to label genomic fragments with distinct genealogical origin at this locus. With the goal to generate haplotype signatures from patients experimentally, we have investigated the suitability of an existing nanofluidic assay platform to perform phase alignment with single-nucleotide polymorphism alleles.
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.
BCL11A is a major regulator of fetal hemoglobin production. Reduced levels of BCL11A have been shown to delay switching from fetal to adult hemoglobin, suggesting that it acts as a stage-specific repressor of gamma globin expression. We have carried out a survey of BCL11A binding in the globin, BCL11A and GATA1 loci by ChIP-on-chip analysis in primary human erythroid cells. We found strong occupancy in both alpha and beta globin upstream regulatory regions as well as in regions involved in switching and hereditary persistence of fetal hemoglobin. Genetic studies have identified a restricted 14kb region in BCL11A intron 2 as being highly associated with HbF levels. Strong GATA-1 binding and acetylated histone H3 was found in this area, which could be indicative of a regulatory element, changes in which might be responsible for the overall regulation of BCL11A. We also observed BCL11A and GATA-1 binding in a known auto-regulatory promoter element of the GATA1 locus.
Sickle cell anemia is one of the commonest causes of stroke in children. It is usually, but not always, associated with intracranial vasculopathy. We have assessed the value of ultrasound screening for extracranial internal carotid artery disease.
Acute Human Parvovirus B19 (HPV B19) infection is the major cause of transient red cell aplasia (TRCA) and acute anaemia in patients with sickle cell disease (SCD). We report three cases of patients who developed nephrotic syndrome (NS) with chronic sequelae after initially presenting with HPV B19-associated TRCA. There was no correlation between evidence of HPV B19 infection and impaired renal function in our cohort of adult sickle cell patients. This is consistent with a view that although NS is potentially a rare complication of symptomatic acute HPV B19 infection, exposure to HPV B19 is not associated with an increased risk of renal disease.
Background Following a clinical evaluation of deferasirox (Exjade) it was concluded that, in addition to baseline body iron burden, ongoing transfusional iron intake should be considered when selecting doses. The 1-year EPIC study, the largest ever investigation conducted for an iron chelator, is the first to evaluate whether fixed starting doses of deferasirox, based on transfusional iron intake, with dose titration guided by serum ferritin trends and safety markers, provides clinically acceptable chelation in patients (aged >or=2 years) with transfusional hemosiderosis from various types of anemia.
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.
Measurements of erythrocytes within the blood are important clinical traits and can indicate various hematological disorders. We report here genome-wide association studies (GWAS) for six erythrocyte traits, including hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). We performed an initial GWAS in cohorts of the CHARGE Consortium totaling 24,167 individuals of European ancestry and replication in additional independent cohorts of the HaemGen Consortium totaling 9,456 individuals. We identified 23 loci significantly associated with these traits in a meta-analysis of the discovery and replication cohorts (combined P values ranging from 5 x 10(-8) to 7 x 10(-86)). Our findings include loci previously associated with these traits (HBS1L-MYB, HFE, TMPRSS6, TFR2, SPTA1) as well as new associations (EPO, TFRC, SH2B3 and 15 other loci). This study has identified new determinants of erythrocyte traits, offering insight into common variants underlying variation in erythrocyte measures.
Sickle cell anaemia (SCA; HbSS) is characterised by its clinical variability, which is only partly explained by known genetic factors. Environmental factors are known to contribute to acute problems but their importance in chronic complications has not been analysed. We have studied 93 children with SCA in a single institution, who underwent transcranial Doppler scanning and steady-state blood tests in 2006. These data were correlated with each individuals exposure to pollution from dust (PM(10)), nitric oxide (NO) and nitrogen dioxide (NO(2)). This exposure was derived from patient postcodes and detailed street-level maps of average pollutant levels in 2006. All the pollutants correlated closely with each other. Increased exposure to pollution correlated with a significant reduction in total bilirubin levels, with a trend towards lower levels of lactate dehydrogenase and aspartate transaminase. There was significant correlation between extracranial internal carotid artery blood velocity and PM(10) exposure. These studies suggest that chronic exposure to air pollutants could explain some variability in SCA. The lower levels of bilirubin and other markers of haemolysis with increased exposure to air pollutants could be mediated by increased exposure to NO.
HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23 is associated with elevated fetal hemoglobin levels and has pleiotropic effects on several hematologic parameters. To investigate potential regulatory activity in the region, we have measured sensitivity of the sequences to DNase I cleavage that identified 3 tissue-specific DNase I hypersensitive sites in the core intergenic interval. Chromatin immunoprecipitation with microarray (ChIP-chip) analysis showed strong histone acetylation in a defined interval of 65 kb corresponding to the core HBS1L-MYB intergenic region in primary human erythroid cells but not in non-MYB-expressing HeLa cells. ChIP-chip analysis also identified several potential cis-regulatory elements as strong GATA-1 signals that coincided with the DNase I hypersensitive sites present in MYB-expressing erythroid cells. We suggest that HMIP contains regulatory sequences that could be important in hematopoiesis by controlling MYB expression. This study provides the functional link between genetic association of HMIP with control of fetal hemoglobin and other hematologic parameters. We also present a large-scale analysis of histone acetylation as well as RNA polymerase II and GATA-1 interactions on chromosome 6q, and alpha and beta globin gene loci. The data suggest that GATA-1 regulates numerous genes of various functions on chromosome 6q.
Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples.
Sickle cell disease (SCD) and beta thalassaemia, caused by lesions that affect the HBB (beta globin gene), form the most common human genetic disorders world-wide, and represent a major public health problem. Inter-individual variation in foetal haemoglobin (HbF) expression is a known and heritable disease modifier; high HbF levels are correlated with reduced morbidity and mortality in both diseases. This review traces our progress in the understanding of the persistence of HbF in adults as a quantitative trait and the genetic approaches used in teasing out the loci contributing to its variability in normal populations and in patients with haemoglobinopathies. Three major loci -- Xmn1-HBG2 single nucleotide polymorphism, HBS1L-MYB intergenic region on chromosome 6q, and BCL11A -- contribute 20-50% of the trait variance in patients with sickle cell anaemia and healthy European Caucasians. It is likely that the remaining trait variance is due to numerous other loci, many contributing modest effects. Identification of the three major loci has not yet been translated into new therapeutic approaches for HbF reactivation but an immediate application would be an improved prediction of ones ability to produce HbF, which in turn, may improve prediction of disease severity.
The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease and myocardial infarction in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.
Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 +/- 0.001 log fL; P < 1.08 x 10(-24)) and PLT (per-G effect -4.55 +/- 0.80 10(9)/L; P < 7.19 x 10(-8)) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis.
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.
Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5 x 10(-7)). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations.
High levels of fetal haemoglobin (HbF) are protective in beta-haemoglobinopathies. The proportion of erythrocytes containing HbF (F-cells, FC) was measured in healthy adults of African and Caucasian ancestry to assess the feasibility of localizing genes for the FC trait using admixture mapping. Participants were Afro-Caribbean (AC) blood donors and residents of a rural enclave with a history of recent German admixture (Afro-German, AG) recruited in Jamaica, and Caucasian Europeans recruited in Jamaica and the UK. FC levels were significantly different between groups (P < 0.001); the geometric mean FC level in the AC sample (n = 176) was 3.75% [95% confidence interval (CI) 3.36-4.18], AG sample (n = 631) was 2.77% (95% CI 2.63-2.92), and among Caucasians (n = 1099) was 3.26% (95% CI 3.13-3.39). After adjustment for age, sex, haemoglobin electrophoresis pattern, and HBG2 genotype, FC levels in the AC group remained significantly different (P < 0.001) from those in the Caucasian and the AG group but the difference between the Caucasian and AG groups became non-significant (P = 0.46) despite substantial differences in average ancestry. The data confirm ethnic differences in FC levels and indicate the potential usefulness of these populations for admixture mapping of genes for FC levels.
Using a genome-wide association study, we found that common inter-individual differences in haemoglobin A(2) (HbA(2) , ?(2) ?(2) ) levels are largely governed by genetic factors (42% of variability). The influence of age (1%) and sex (4%) was small. HbA(2) levels were influenced by two loci: the HBS1L-MYB locus on chromosome 6q, which has been shown to have pleiotropic effects on other haematological traits; and a second locus surrounding HBB, the gene encoding ?-globin. Our results suggest that HbA(2) levels in adults are influenced by two different biological processes: one via kinetics of erythropoiesis, and the other, via competition between HBB and HBD activity.
We describe a novel deletion causing ???? thalassemia in a Pakistani family. The Pakistani deletion is 506kb in length, and the second largest ???? thalassemia deletion reported to date. It removes the entire ? globin gene (HBB) cluster, extending from 431kb upstream to 75kb downstream of the ? globin gene (HBE). The breakpoint junction occurred within a 160bp palindrome embedded in LINE/LTR repeats, and contained a short (9bp) region of direct homology which may have contributed to the recombination event. Characterization of the deletion breakpoints has been particularly challenging due to the complexity of DNA deletion, insertion and inversion, involving a multitude of methodologies, mirroring the changing DNA analysis technologies.
Patients with beta (?)-thalassemia who have high levels of fetal hemoglobin (HbF) have less severe anemia and are often transfusion-independent. Therefore, augmentation of HbF production has been a longstanding therapeutic objective. Three classes of HbF-inducing agents have been investigated for the treatment of ?-thalassemia including chemotherapeutics, short-chain fatty acid derivatives, and recombinant erythropoietin. These agents have several different mechanisms of action and have been shown to increase total hemoglobin levels by 1-5 g/dL above baseline, but none has been able to sustain the therapeutic levels needed to maintain transfusion independence. Recent findings have provided new insights regarding HbF regulatory pathways, providing new opportunities for derepression of fetal globin gene expression and HbF induction.
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