David H.K. Chui

Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies

HbVar (http://globin.cse.psu.edu/globin/hbvar/) is a relational database developed by a multi-center academic effort to provide up-to-date and high quality information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Extensive information is recorded for each variant and mutation, including sequence alterations, biochemical and hematological effects, associated pathology, ethnic occurrence and references. In addition to the regular updates to entries, we report two significant advances: (i) The frequencies for a large number of mutations causing beta-thalassemia in at-risk populations have been extracted from the published literature and made available for the user to query upon. (ii) HbVar has been linked with the GALA (Genome Alignment and Annotation database, available at http://globin.cse.psu.edu/gala/) so that users can combine information on hemoglobin variants and thalassemia mutations with a wide spectrum of genomic data. It also expands the capacity to view and analyze the data, using tools within GALA and the University of California at Santa Cruz (UCSC) Genome Browser.

Prenatal exclusion of β thalassaemia major by examination of maternal plasma

Summary The discovery of the presence of fetal DMA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of p thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (–CTTT) mutation in the β globin gene from maternal plasma by real-time PCR. The specificity and sensitivity of the allele-specific assay was confirmed by subjecting plasma, buffy coat, and amniotic fluid samples from 100 pregnancies to screening for the mutation. Subsequently, the assay was applied to the prenatal testing of eight fetuses at risk of β thalassaemia major, the aim being to exclude fetal inheritance of paternally transmitted COdon 41/42 mutation. The fetal genotype was completely concordant with conventional analysis and β thalassaemia major was excluded in two of the pregnancies non-invasively.

Fetal hemoglobin in sickle cell anemia

Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Some patients with sickle cell disease have exceptionally high levels of HbF that are associated with the Senegal and Saudi-Indian haplotype of the HBB-like gene cluster; some patients with different haplotypes can have similarly high HbF. In these patients, high HbF is associated with generally milder but not asymptomatic disease. Studying these persons might provide additional insights into HbF gene regulation. HbF appears to benefit some complications of disease more than others. This might be related to the premature destruction of erythrocytes that do not contain HbF, even though the total HbF concentration is high. Recent insights into HbF regulation have spurred new efforts to induce high HbF levels in sickle cell disease beyond those achievable with the current limited repertory of HbF inducers.

Prevalence and Genotypes of α- and β-Thalassemia Carriers in Hong Kong — Implications for Population Screening

Background The thalassemias are common in southern China. We determined the prevalence of heterozygous carriers of these genetic disorders in Hong Kong and assessed the feasibility of a community-based screening program. Methods An educational and screening program for the thalassemias was carried out in three high schools with a total of 2420 students. Seventy-five percent of the students agreed to undergo screening, which consisted of blood counts, hemoglobin electrophoresis, serum ferritin measurements, and DNA analyses. Results Of the 1800 blood samples tested, 150 (8.3 percent) had microcytosis (mean corpuscular volume, <80 μm3). Ninety students (5.0 percent) were carriers of α-thalassemia, of whom 81 (4.5 percent) were carriers of the Southeast Asian type of deletion, in which both α-globin genes on the same chromosome 16 are deleted. Sixty-one students (3.4 percent) were carriers of either β-thalassemia or the mutation coding for hemoglobin E. Six students were carriers of both α- and β-thalassemia...

BCL11A is a major HbF quantitative trait locus in three different populations with β-hemoglobinopathies ☆

Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of beta-thalassemia major and sickle cell anemia. Recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with beta-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with beta-thalassemia trait, and with HbF levels in Thais with either beta-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.

Fetal hemoglobin in sickle cell anemia: genome-wide association studies suggest a regulatory region in the 5′ olfactory receptor gene cluster

In a genome-wide association study of 848 blacks with sickle cell anemia, we identified single nucleotide polymorphisms (SNPs) associated with fetal hemoglobin concentration. The most significant SNPs in a discovery sample were tested in a replication set of 305 blacks with sickle cell anemia and in subjects with hemoglobin E or beta thalassemia trait from Thailand and Hong Kong. A novel region on chromosome 11 containing olfactory receptor genes OR51B5 and OR51B6 was identified by 6 SNPs (lowest P = 4.7E-08) and validated in the replication set. An additional olfactory receptor gene, OR51B2, was identified by a novel SNP set enrichment analysis. Genome-wide association studies also validated a previously identified SNP (rs766432) in BCL11A, a gene known to affect fetal hemoglobin levels (P = 2.6E-21) and in Thailand and Hong Kong subjects. Elements within the olfactory receptor gene cluster might play a regulatory role in gamma-globin gene expression.

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

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.

Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro

Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES) cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells, but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.

Universal Newborn Screening for Hb H Disease in California

Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Barts (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Barts levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Barts as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Barts hydrops fetalis syndrome.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.