We present a patient with a compound heterozygosity codon 39 (C>T) ((0)) [or 39(C5)GlnStop (G39X); CAG>TAG; HBB: c.118C>T] and -87 (C>T) ((+)) (HBB: c.-137C>T) -globin mutations, a non transfusion-dependent thalassemia phenotype and 97.0% fetal hemoglobin. A novel heterozygous mutation was identified in a highly conserved residue in the COOH-terminus of the Kruppel-like factor 1, R360H, that likely altered DNA-binding and impaired transactivation.

Hereditary persistence of fetal hemoglobin (HPFH) and (δβ)0 thalassemia are caused by deletions within the β-globin gene (HBB) cluster that remove elements that affect the expression of the γ-globin genes (HBG2 and HBG1, or HBG). These deletions are of different lengths and have different 5’ and 3’ breakpoints. The phenotypes associated with heterozygous carriers of (δβ)0 thalassemia and HPFH deletions are differentiated by levels of 5-15% HbF distributed heterocellularly in the former and 15-30% HbF distributed pancellularly in the latter. We found a novel 588.6 kb deletion that removed both the 3.5 kb fragment 5’ to HBD that is deleted in Corfu β thalassemia and contains a BCL11A binding site, and the known cis-acting elements downstream of HBB. The proband with this deletion had a HbF of 5.4% (Morrison et al, Blood, 2014 abstract 3452). To study the relative importance of 5’ and 3’ regulatory elements in HBG expression we studied 209 cases culled from the literature and from our laboratory where the 3.5 kb element 5’ to HBD and enhancers 3’ to HBB were deleted and HBG remained intact. We used a backwards stepwise regression statistical analysis to determine which deleted elements had the greatest effect on HbF levels. The combination of the deletion of 3.5 kb intergenic region 5’ to HBD, the presence of the HPFH-1 “3D” enhancer juxtaposed to HBG, and the deletion of the 3’ HS1 region accounted for 66.7% of the HbF variation in heterozygotes for HPFH and (δβ)0-thalassemia deletions. The HPFH-1 “3D” enhancer juxtaposed to HBG— the main difference between HPFH-1 and 2 compared with Spanish (δβ)0-thalassemia—was associated with an increase in HbF of 20.78% (p<2e-16) after adjusting for the effects of the other 5’ and 3’ cis-acting elements. The next most significant factor was the deletion of the 3.5 kb fragment 5’ to HBD which resulted in an increase of 10.62% HbF after similar adjustments (p<2e-16); deletion of the 3’ HS1 region accounted for an increase in HbF of 5.25% (p<1.05e-5). The HPFH-3 and HPFH-6 enhancer regions each accounted for a less than 1% increase in HbF and were not significantly associated with HbF in this model. Among 194 individuals where both 5’ and some 3’ elements affecting γ-globin gene expression—excluding the “3D” enhancer—were deleted, HbF was 20±9.3%; in 13 cases where all 3’ enhancers—including the “3D” enhancer—were deleted, HbF was 6.8±3.7% (p=8.9e-07). To determine which combinations of cis-acting elements were associated with high and low HbF levels we performed a classification and regression tree (cART) analysis on HbF. The results of the regression tree (Figure) only included the deletion of the 5’ 3.5 kb fragment region, the presence of the HPFH-1 “3D” enhancer and the deletion of the 3’ HS1 region and were consistent with the results of the backwards selection model. The absence of the 5’ 3.5 kb fragment 5’ to HBD combined with the presence of the HPFH-1 “3D” enhancer was associated with the highest average HbF of 27.02%. The absence of the 3.5 kb fragment 5’ to HBD combined with the absence of the HPFH-1 “3D” enhancer was associated with the lowest average HbF of 6.82%.The 588.6 kb deletion is the largest deletion reported in the HBB cluster that leaves the γ-globin genes intact, and the second to remove both the BCL11A binding site and all known 3’ enhancer elements. By studying deletions in the HBBgene cluster we have further defined the hierarchy of cis-acting elements that modulate HbF levels in adults and suggest a paramount role of the distal “3D” enhancer.

Abstract 1030 This icon denotes a clinically relevant abstract

Objective. Study the effect of loss of expression of Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, on hematopoietic stem cells (HSCs).Methods. We examined the fetal livers of mouse embryos with homozygous disruption of the Pitx2 gene, using flow cytometry immunophenotyping analysis, as well as immunohistochemistry techniques. We further investigated the role of Pitx2 in HSCs using a chimeric mouse model system. Pitx2 null embryonic stem (ES) cell clones were generated from embryonic day 3.5 blastocysts of Pitx2 null embryos. The Pitx2 null donor ES cell contribution to the adult hematopoietic system was confirmed by identifying donor-specific glucose-phosphate isomerase isotype in the erythrocytes using cellulose acetate eletrophoresis, and by demonstrating donor-specific major histocompatibility complex antigen allotype on the granulocytes/monocytes and T and B lymphocytes of the chimeric mice using flow cytometry analysis.Results. Pitx2 homozygous null fetal livers are decreased in size and overall cellularity. The erythroid cell component of these livers is further reduced as compared to that of their wild-type and heterozygous littermates. Detailed quantitative analysis of the chimeric mice revealed contribution of Pitx2 null ES cells to erythroid, myeloid, lymphoid, and megakaryocytic lineages. The quantitative level of ES cell contribution to the peripheral hematopoietic cells was proportional to the level of general chimerism as determined by coat color.Conclusion. Although the fetal livers of Pitx2 null embryos displayed signs of impaired erythropoiesis, Pitx2 gene disrupted HSCs can contribute to hematopoiesis under physiological conditions. (C) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Alpha thalassemia is a genetic disorder of hemoglobin production that typically is inherited in an autosomal co-dominant fashion. Rare forms of alpha-thalassemia, however, occur as de novo or acquired disorders. These disorders occur in two clinical situations: 1) alpha-thalassemia associated with mental retardation, and 2) acquired alpha-thalassemia (HbH disease) associated with myelodysplastic syndrome. Study of these rare disorders has led to the identification and characterization of a gene on the X chromosome (called ATRX) that encodes a trans-acting factor capable of influencing the expression of alpha-globin and other genes.

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3' end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from "stem cell genes" being highly expressed at 0 h and turned off at 48 h, while "cell division" genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

Objective. Hematopoietic stem cells (HSCs) represent a rare and incompletely characterized fraction of marrow cells that are capable of both self-renewal and differentiation into all of the mature cells in the peripheral blood, We undertook to identify genes expressed preferentially by HSCs as an initial step toward better understanding the molecular mechanisms that underlie HSC behavior.Methods, We modified the representational difference analysis technique to isolate gene fragments present in amplified cDNA prepared from highly purified murine hematopoietic stem/ progenitor cells (Lin(-)/Hoechest(low)/rhodamine(low)) and absent (or much less abundant) in amplified cDNA prepared from lineage-committed marrow cells. We went on to use one potentially important gene fragment that we isolated in this way, to screen a cDNA library prepared from these cells and to characterize the pattern of expression of the gene in hematopoietic and other cells.Results. We isolated a fragment of the homeobox transcription factor Pitx2, from amplified cDNA prepared from murine hematopoietic stem/progenitor cells. From a cDNA library prepared from these cells, a full-length cDNA was isolated that corresponds to one of the three known isoforms of Pitx2 (Pitx2c), Pitx2c is expressed in murine embryonic stem (ES) cells and in hematopoietic stem/progenitor cells but not in more differentiated hematopoietic cells or in a large panel of established murine hematopoietic cell lines. Pitx2c expression was not detected after 48 hours of in vitro cytokine stimulation of hematopoietic stem/progenitor cells,Conclusions. Pitx2c is expressed in hematopoietic stem/progenitor cells but not in their differentiated progeny. The pattern of expression of Pitx2c in primitive hematopoietic stem/progenitor cells suggests that it may play a role in hematopoietic stem-cell biology. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.

Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1 chromosomal gene. Characterization of theANK-1 gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over ∼160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the “hinge” region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of theANK-1 gene.

The human erythrocyte β-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid β-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the β-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present β-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat β10 is encoded by 4 exons. No single position of an intron in the β-spectrin gene is conserved between any of the 17 β-spectrin and 22 α-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5′ donor splice-site sequence begins with GC. The β-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid β-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp.

A 96-bp synthetic oligonucleotide corresponding to an amino acid sequence near the N-terminus of erythroid β-spectrin was used to screen a human genomic library, and two overlapping recombinants were isolated. DNA sequence analysis established that the genomic fragment encoded β-fodrin, the nonerythroid form of β-spectrin, by correlation to a known amino acid sequence of human brain β-fodrin. The genomic DNA contained regions that cross-hybridized with an erythroid β-spectrin cDNA probe, and the DNA sequence of these regions revealed a high degree of identity with that of erythroid β-spectrin and a similar exon/intron organization. A single-copy DNA fragment of the β-fodrin genomic clone was used to screen a lymphoid cell cDNA library and two recombinants were isolated. The composite DNA sequence of these various genomic and cDNA clones encoded almost all of the first twelve 106 amino acid repeat segments of β-fodrin that shared 58% identity and 75.5% similarity with the amino acid sequence of β-spectrin and 66% identity with the nucleotide sequence of β-spectrin cDNA. The chromosomal localization of the gene was determined to be chromosome 2 by hybridization of a single-copy probe derived from the cloned genomic DNA to DNA of a panel of somatic hybrid cell lines, and in situ hybridization localized the gene to band 2p21. β-Fodrin was assigned the gene symbol SPTBN1 .

Erythropoietin (EPO) mediates the growth and differentiation of erythroid progenitors through its interaction with a specific receptor. Using a partial cDNA clone for the murine erythropoietin receptor, we isolated a human genomic clone containing the erythropoietin receptor gene. The coding region of the human EPO receptor gene is contained within eight exons spanning approximately 6 kb. The human gene has a great deal of structural similarity and sequence homology with the murine gene. The murine gene also has eight exons, although the size of each intron is somewhat different. The locations at which the introns interrupt the coding sequence are conserved precisely. The genomic organization of the EPO receptor gene is also shown to be homologous to the genomic organization of the IL-2 receptor beta chain gene. The sequence of 1.1 kb of 5' flanking DNA was characterized and contains consensus sequences for both Sp1 and GATA-1 binding sites and an initiator (Inr)-like element, but lacks both a canonical TATA box and the CACCC consensus sequence found in the murine gene.

HEREDITARY spherocytosis is a heterogeneous disorder characterized by hemolytic anemia, spheroidal red cells, and increased osmotic fragility of erythrocytes. The majority of the patients have an a...

Hybrids formed between human α and β globin cDNA and total human cellular DNA have been studied by thermal denaturation and cesium chloride density gradient centrifugation. From these studies, the weight average G+C content of human α globin cDNA has been determined to be 62%±2% and that of human β globin cDNA 51%±2%. These values correlate well with the results of G+C content of the human α and β globin cDNAs as determined by direct nucleotide sequence analysis of the cDNAs. Thermal denaturation and cesium chloride density gradient centrifugation of DNA-cDNA hybrids can therefore provide accurate information on the base composition of mRNA related sequences of any single copy gene for which a relatively pure cDNA can be obtained, without the necessity for direct nucleotide sequence analysis.

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.