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Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as α-globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

In higher eukaryotes, many genes are regulated by enhancers that are 104–106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7–22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding. Micro Capture-C allows physical contacts to be determined at base-pair resolution, revealing that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters.

Mammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ∼70 kb sub-TAD containing the duplicated mouse α-like globin genes and their five enhancers (5’-R1-R2-R3-Rm-R4-α1-α2-3’). The 5’ border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the α-globin locus leaves the sub-TAD largely intact. The predominant 3’ border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed α2-globin gene rather than the CTCF sites at the 3’-end of the sub-TAD. Of interest, the almost identical α1- and α2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal α1-globin gene which behaves as a partial boundary between the enhancers and the distal α2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements.Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34+ progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal ‘spongy’ chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41, as compared to the other known causative gene CDAN1, indicating a genotype/phenotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia.

β-Thalassaemia is one of the most common monogenic diseases with no effective cure in the majority of patients. Unbalanced production of α-globin in the presence of defective synthesis of β-globin is the primary mechanism for anaemia in β-thalassaemia. Clinical genetic data accumulated over three decades have clearly demonstrated that direct suppression of α-globin and induction of γ-globin are effective in reducing the globin chain imbalance in erythroid cells hence improving the clinical outcome of patients with β-thalassaemia. Here, we show that the histone deacetylase inhibitor drug, vorinostat, in addition to its beneficial effects for patients with β-thalassaemia through induction of γ-globin, has the potential to simultaneously suppress α-globin. We further show that vorinostat exhibits these synergistic beneficial effects in globin gene expression at nanomolar concentrations without perturbing erythroid expansion, viability, differentiation or the transcriptome. This new evidence will be helpful for the interpretation of existing clinical trials and future clinical studies that are directed towards finding a cure for β-thalassaemia using vorinostat.

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k-mer-based analysis of DNase footprints to determine any k-mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

PhenCode (Phenotypes for ENCODE; http://www.bx.psu.edu/phencode) is a collaborative, exploratory project to help understand phenotypes of human mutations in the context of sequence and functional data from genome projects. Currently, it connects human phenotype and clinical data in various locus-specific databases (LSDBs) with data on genome sequences, evolutionary history, and function from the ENCODE project and other resources in the UCSC Genome Browser. Initially, we focused on a few selected LSDBs covering genes encoding alpha- and beta-globins (HBA, HBB), phenylalanine hydroxylase (PAH), blood group antigens (various genes), androgen receptor (AR), cystic fibrosis transmembrane conductance regulator (CFTR), and Bruton's tyrosine kinase (BTK), but we plan to include additional loci of clinical importance, ultimately genomewide. We have also imported variant data and associated OMIM links from Swiss-Prot. Users can find interesting mutations in the UCSC Genome Browser (in a new Locus Variants track) and follow links back to the LSDBs for more detailed information. Alternatively, they can start with queries on mutations or phenotypes at an LSDB and then display the results at the Genome Browser to view complementary information such as functional data (e.g., chromatin modifications and protein binding from the ENCODE consortium), evolutionary constraint, regulatory potential, and/or any other tracks they choose. We present several examples illustrating the power of these connections for exploring phenotypes associated with functional elements, and for identifying genomic data that could help to explain clinical phenotypes.

Summary Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Two Spanish families with α thalassaemia, including 4 individuals with Hb H disease, are described. DNA mapping shows that, in addition to the common α thalassaemia determinant (-α3.7), a different and previously unreported allele is present in each family. In one, there is a deletion of 10.5–12 kb of DNA including both α genes (-SPAN). in the other, a deletion of more than 100 kb has removed the entire α globin gene complex (-BR).

Restriction endonuclease analysis has been performed on the alpha and beta globin gene clusters of 57 Cypriots homozygous for beta thalassaemia, 30 with the transfusion dependent form of the condition (thalassaemia major) and 27 who are less severely affected (thalassaemia intermedia). There was a significant difference in the incidence of alpha thalassaemia between the two groups: 14/27 of the patients with thalassaemia intermedia also had deletion forms of alpha thalassaemia, while only 4/30 of the patients with thalassaemia major were similarly affected. Thus in Cypriot patients who are homozygous for beta thalassaemia the co-inheritance of alpha thalassaemia is an important factor in determining the clinical course.

Over the past 50 years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the alpha- and beta-globin genes are normally regulated and documenting the effects of inherited mutations that cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found more often. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression that, in turn, may open up new avenues for improving the management of patients with common types of thalassemia.

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching. © 2003 Elsevier Science (USA). All rights reserved.

Our understanding of biological processes in humans is often based on examination of analogous processes in other organisms. The nematode worm Caenorhabditis elegans has been a particularly valuable model, leading to Nobel prize winning discoveries in development and genetics. Until recently, however, the worm has not been widely used as a model to study transcription due to the lack of a comprehensive catalogue of its RNA transcripts. A recent study by Chen et al. uses next-generation sequencing to address this issue, mapping the transcription initiation sites in C. elegans and finding many unexpected similarities between the transcription of enhancers and promoters in the worm and mammalian genomes. As well as providing a valuable resource for researchers in the C. elegans community, these findings raise the possibility of using the worm as a model to investigate some key, current questions about transcriptional regulation that remain technically challenging in more complex organisms.

DNA methylation is an epigenetic modification that plays an important role in development and differentiation. The patterns of DNA methylation are largely established in early embryogenesis and maintained during development. Abnormal DNA methylation patterns have been associated with many human diseases, including cancer. Despite its importance, little is currently known about the mechanisms that determine DNA methylation patterns throughout the genome. To shed light on the molecular mechanisms that regulate DNA methylation, this study investigates whether DNA methylation patterns are established and maintained normally when human DNA is placed into a heterologous murine environment as opposed to its natural, endogenous chromosomal environment. Here, a previously generated transgenic mouse model, containing 117 kb of human DNA bearing the human α-globin cluster and all of its known regulatory elements, was analysed. The pattern of DNA methylation of the endogenous human α-globin cluster was compared with that of the transgenic cluster in the background of mouse embryonic stem cells (ESCs) and tissues. It was found that, although the normal human DNA methylation pattern was largely established and maintained in a mouse background, the region immediately around the human α-globin genes themselves is generally less methylated in mouse compared to human ESCs. It was found that regions adjacent and up to 2kb from the CpG islands (CGIs), so-called CGI shores, were unusually hypomethylated: this seems to be the result of an extension of CGIs in humanised mouse (hm) ESCs compared to human (h) ESCs. Furthermore, this hypomethylation appeared to increase during development in both erythroid and non-erythoid cells. To identify any cis-regulatory sequences responsible for the hypomethylated state of human CGI shores in the mouse, 2-4 kb human test sequences containing the CGI associated with the human α-globin 2 (α2) gene and its adjacent hypomethylated shore were re-integrated into the mouse α-globin locus via recombination-mediated cassette exchange (RMCE). Human CGI shores became hypomethylated in the context of the re-integrated test sequences, indicating that the appearance of hypomethylation is determined by the underlying human DNA sequence in the test fragments. In summary, the data presented here reveal that human CGIs become extended when placed in a mouse background leading to hypomethylation of human CGI shores in the mouse compared to the pattern of methylation at the normal endogenous human locus. These findings suggest that species-specific factors determine DNA methylation near CGIs. The transgenic mouse model provides an excellent system to dissect out species-specific regulation of CGI shore methylation. Furthermore, this study lays the foundation for future experiments addressing the role of DNA methylation in regulating human gene expression in the murine context, and examining the validity of transgenic mouse models for the study of human gene regulation.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

ATRX syndrome is characterized by X-linked mental retardation associated with α-thalassemia. The gene mutated in this disease, ATRX , encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: ( i ) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; ( ii ) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; ( iii ) in extract from cells of a patient with ATRX syndrome, the level of the Daxx–ATRX complex is correspondingly reduced; ( iv ) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and ( v ) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

The terminal regions of chromosomes have specialised molecular and cytological properties, which are reflected in their DNA sequence organisation. Telomeric DNA, the conserved sequence at the extreme ends of chromosomes, comprises in mammals a variable length (2-20 kb in humans; 15-150 kb in mouse) of the simple sequence (TTAGGG)n orientated 5’->3’ toward the chromosome end. It is likely that this repeat motif, together with associated protein components can explain many of the properties of human telomeres; for example, replication of the terminal DNA, protection against exonucleolytic degradation, and the prevention of chromosome fusion (reviewed in Blackburn, 1991).

A rapid and inexpensive polymerase chain reaction (PCR) based strategy is described which detects the three common, severe alpha thalassaemia determinants observed in southeast Asia (--SEA) and the Mediterranean (--MED and -(alpha)20.5). Oligonucleotide primers have been chosen which allow specific identification of both normal (alpha alpha) and abnormal (--) chromosomes using identical conditions in either the same or parallel PCR reactions. This strategy should be useful in the development of screening programmes to identify carriers of alpha thalassaemia (--/alpha alpha) and prenatal diagnosis of the Hb Bart's hydrops fetalis syndrome (--/--) for those populations in which this represents a major cause of perinatal death.

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.

Introduction: In contrast to the classical thalassemias, two distinct thalassemias were recently described in which the molecular defect does not reside in the globin genes but in a transcriptional activator of alpha-globin genes. This protein, ATRX, is mutated in the rare inherited disease of alpha-thalassemias (AT) with mental retardation (ATR-X syndrome) whose affected individuals show a mild form of AT. In addition, and independent of the ATR-X syndrome, there have been approximately 100 case reports worldwide of the association of an acquired form of AT with hematological neoplasms, the large majority of those cases being MDS (ATMDS). The clinical characteristics of such patients encompass the typical features of the underlying hematological disorder plus microcytic anemia. The latter is due to massively reduced alpha-globin gene transcription resulting in excess hemoglobin H (HbH), as revealed by supravital staining of peripheral blood erythrocytes and hemoglobin electrophoresis. The molecular defect of ATMDS lies in a mutation of the ATRX protein and, thus, represents a form of acquired alpha-thalassemia. ATMDS shows a striking male preponderance for reasons that are as yet unclear. We systematically studied patients with MDS and low MCV and/or MCH but without iron deficiency for the possible presence of HbH cells (indicative of ATMDS). Supravital staining and sequence analysis of the ATRX gene were performed as described (Steensma et al. Blood.2004;103(6):2019–26). Results: Two female pts with AT-MDS were identified by this strategy. Pat 1 was a 69 year old pt who diagnosed with MDS of the subtype refractory cytopenia with multilineage dysplasia (RCMD), ringed sideroblasts and thrombocytosis (MDS/MPS overlap syndrome, JAK2 negative). She presented with microcytic anemia (Hb 7,5 g/dl, MCV 76,5 fl, MCH 22.4 pg). Supravital staining of a peripheral blood smear revealed massive erythrocytic HbH inclusions. Sequencing of the ATRX coding sequence revealed a novel missense mutation with an A>G transition in codon 2234. This mutation (D2234G) results in an amino acid substitution in ATRX exon 32. It represents the 14th ATRX mutation described thus far and, moreover, the first mutation detected in a female. Pat 2 (61 years old) with initially RCMD and microcytic anemia (Hb 10.4 g/ld, MCV 69 fl, MCH 15.2 pg) had increasing erythrocytosis of 6.93 Mio/μl maximum (also JAK2 negative). Molecular analysis of the ATRX gene also showed a not previously reported ATRX point mutation in exon 8 (G521A) which results in an amino acid change from cysteine to tyrosine and consequently in loss of a zinc finger. Conclusions: Though AT-MDS is mostly diagnosed in males we have identified two female patients, both showing novel ATRX somatic mutations. ATMDS might be more frequent in female than previously thought. Microcytic anemia in association with a hematological neoplasm, most commonly MDS, should alert to ATMDS which is easily diagnosed by supravital staining of peripheral blood smears. The remarkable thrombocytosis and erythrocytosis, respectively, in our 2 pts are at least suggestive of other phenotypic abnormalities possibly associated with the acquired ATRX genotypes on the MDS background.

The basic Helix-Loop-Helix (bHLH) transcription factor SCL is required for specification of haematopoietic stem cells (HSCs) and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. We challenged this concept by showing that SCL DNA-binding activity was dispensable for some of its functions using SCL−/− ES cells rescued with a DNA-binding defective SCL mutant (SCL-RER) (Porcher et al. Development,1999). We have now studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line SCL-RER mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of primitive erythroid progenitors was observed in SCL RER/RER mutant yolk sacs. At day E12.5 and E13.5, homozygote mutant SCL RER/RER embryos were smaller and paler than wild-type (wt) and heterozygote littermates but presented at the expected mendelian frequency. Lethality was first observed at day E14.5. However, 7% of homozygote mice were born from heterozygous crosses. Surviving adult mice presented with mild microcytic hypochromic anemia. Assessment of progenitor replating potential showed qualitative defects with poor haemoglobinisation of homozygote-derived fetal and adult CFU-Es compared to controls. To understand the cause of the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for either early progenitors or late normoblasts. We observed decreased or increased mRNA expression of most genes tested in mutant-derived erythroid populations when compared to controls matched for differentiation stage, thereby showing that SCL DNA-binding activities are required for both activation and repression of target genes. As examples, mRNA for red cell membrane protein Band 4.2 was dramatically decreased; in contrast, alpha-globin expression was up-regulated in early progenitors, indicating that SCL DNA-binding activity might be required for repression of alpha-globin levels in this setting. We then pursued the analysis of SCL-mediated alpha-globin gene regulation by chromatin immunoprecipitation (ChIP) analysis and tested 4 DNase I hypersensitive sites (DHS) previously shown to bind SCL. From material derived from mutant fetal liver cells, we observed slight variations of SCL binding on 3 out of the 4 cis-acting elements when compared to controls. Importantly, there was a dramatic decrease in SCL binding on the fourth DHS site (HS-12). We concluded that SCL DNA-binding activity was likely to be directly required for repression of alpha-globin levels in erythroid progenitors. Interestingly, we have recently characterised the interaction of SCL with a co-repressor complex comprising the oncoprotein ETO-2 in erythroid cells. We have now shown by ChIP that ETO-2 occupies the alpha-globin locus on HS-12 in wt erythroid progenitors, but not in more mature cells, therefore suggesting a role for the SCL/ETO-2 complex in repression of erythroid-specific genes in the early stages of erythroid maturation. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of HSCs and allows characterisation of DNA-binding requirements throughout development. Combined with studies of the dynamic of SCL-containing multiprotein complexes during erythroid maturation, this model will help define the molecular pathways involved in erythropoiesis.

Although a great deal is known about the molecular mechanism underlying α thalassemia (thal), which is characterized by reduced or absent α-globin expression, the molecular basis of α thalassemia in Pacific Islanders is not fully understood. Previously single α gene deletion alleles (−α3.7 and −α4.2) were the only types of α thalassemia identified in this population, however these genotypes do not account for several cases of Hb H disease in the Melanesian Islanders of Papua New Guinea (PNG) and Vanuatu. To date, no mutations have been found in the α globin gene cluster of these individuals. To determine the cause of α thalassemia in this population, we studied 35 individuals from three unrelated families of Pacific Islanders. 16 of them had classical Hb H disease characterized by mild to moderate anemia (Hb; 10.3 ± 2.3 g/dL), microcytosis (MCV; 66 ± 3.5 fL), hypochromia (MCH; 19.8 ± 1.6 pg), positive Hb H by electrophoresis (5–18%) and numerous Hb H inclusion bodies in peripheral blood. Gene mapping revealed that six had normal genotypes (αα/αα) while the rest were heterozygotes for α+ thalassemia (−α/αα). Preliminary sequence analyses demonstrated that the α globin genes and their regulatory elements were entirely normal casting some doubt on whether this novel form of α thal is genuinely linked to chromosome 16. Using a combination of a long range haplotype analysis by MALDI-TOF to simultaneously genotype 80 SNPs covering 350 kb of the telomeric end of chromosome 16 (16p; 13.3) and several tandem repeat markers (VNTRs and HVRs), we demonstrated that the α thal phenotype is strongly associated with a single haplotype (IIIa;PNG) found in these cases. Consistent with the Hb H patients, individuals from these families and another 15 unrelated individuals (4.7%) identified in a population survey (n = 315) who were hypochromic (MCH < 25 pg), despite having normal α genes (αα/αα) and iron status, were also found to carry the same type of chromosome 16 linked to the affected haplotype. These findings indicate that this form of α thalassemia is unequivocally linked to chromosome 16. Subsequent linkage disequilibrium analysis (LD) in these cases limited the chromosomal interval containing the mutation to within 169 kb from the 16p-telomere. Extensive genomic mapping using more than 10 restriction enzymes and 37 plasmid and PCR probes together with Pulse Field Gel Electrophoresis (PFGE), covering 400 kb of 16p, excluded small deletions, insertions, rearrangements and chromosomal truncations in the affected segment of chromosome 16. Direct genomic sequencing of collectively 40 kb of the α globin genes, all known multispecies conserved sequences (MCS-R, 1–4) demonstrated no apparent mutation. Interestingly, interspecific somatic cell hybrid analysis of the affected chromosome 16 in a mouse erythroid leukemia (MEL) background did not recapitulate the phenotype of α thalassemia. These results suggest a novel mechanism, either at the genetic or epigenetic level, underlying the reduced expression of the α globin genes in the Pacific Islanders and understanding such mechanism might provide more insights on the complexity of stage- and tissue-specific gene regulation in human erythroid environment.

We have studied the interaction of the alpha alpha alpha/alpha alpha gene arrangement with various beta globin genotypes (AA, AS, AC, SS and SC). Whereas this interaction has no detectable clinical or haematological effects in subjects with AA, SS or SC genotypes it is associated with a significantly increased level of Hb S or Hb C in heterozygotes for these variants. These findings indicate that the additional alpha globin gene in the alpha alpha alpha gene arrangement is functional.

We have studied seven Jamaican Negro families in whom the genes for alpha thalassaemia and the sickle cell mutation (betas) were independently segregated. Using a combination of techniques we identified two alpha thalassaemia phenotypes which resemble the severe (alpha thalassaemia 1) and mild (alpha thalassaemia 2) determinants previously described in Orientals. This study has enabled us to clearly correlate the phenotype of alpha thalassaemia with the genotype in this population. Furthermore, since in each family alpha thalassaemia was present in association with the gene for the sickle cell mutation we have determined the proportion of Hb S in the peripheral blood of individuals with the alpha alpha/alpha alpha, -alpha/alpha alpha and -alpha/-alpha genotype who are also heterozygous for the betas mutation. Genetic analysis in these families shows that in each case subjects with the alpha thalassaemia 1 phenotype are homozygous for the alpha thalassaemia 2 defect (-alpha/-alpha). We have found no instances of the genotype --/alpha alpha in this population which may explain the rarity of the severe alpha thalassaemia syndromes in Jamaica. Restriction mapping data in the alpha thalassaemia 2 homozygotes from this population shows that the (-alpha/) haplotype results from a deletion of one of the linked pair of alpha globin genes and that this has probably arisen by an unequal crossover between non-homologous alpha genes.

A combination of polymorphic DNA markers, cytogenetic analysis, and in situ hybridisation has been used for the high resolution assignment of the human alpha globin gene cluster on chromosome 16. Multiallelic DNA probes from within the alpha globin cluster were used to determine the number of copies of this locus in three cell lines containing trisomies of the short arm of chromosome 16 and one with a familial inversion, inv(16). The breakpoints in these rearrangements flank the alpha globin locus and locate a shortest region of overlap to 16p13.1. A meiotic crossover was also localised to this band. In situ hybridisation of biotinylated DNA probes to normal and inverted chromosomes 16 [inv(16)(p13.1;q22)] showed hybridisation sites at opposite ends of the chromosomes, consistent with this regional localisation.

Three identical alpha + thalassemia genes, one of which always carried the Hb J Tongariki mutation, have been observed in Vanuatuans. Despite the fact that at least two of them have arisen by different types of crossover event, the expression of all three haplotypes is identical.

We have characterised a cluster of CpG rich (HTF) islands in the alpha-globin complex and report here two unusual features: The human embryonic zeta 2-globin gene is associated with an HTF island within its 3' portion rather than at the 5' end. Furthermore at least two non-functional pseudogenes within the cluster (psi zeta 1 and psi alpha 2) are associated with CpG rich islands.

A study of rheological determinants (plasma viscosity, whole-blood viscosity, and erythrocyte deformability) was made in 24 matched pairs of patients with homozygous sickle cell disease, with and without homozygous alpha-thalassaemia 2. Patients with coexisting alpha-thalassaemia showed a significant increase in erythrocyte deformability measured as filtration of washed erythrocytes through 5 micron diameter pores and also as viscosity of whole blood at high shear rate (230s-1) and standard haematocrit (0.45). This rheological advantage may explain the beneficial effect of alpha-thalassaemia 2 on haematological parameters and clinical events in homozygous sickle cell disease.

Studies in two Jamaican Negro families, including haematological and haemoglobin analysis, haemoglobin synthesis, and globin messenger-RNA assay, have defined two alpha-thalassaemia phenotypes which resemble the severe (alpha-thalassaemia 1) and mild (alpha-thalassaemia 2) forms of the disorder described in Orientals. Genetic analysis suggests that subjects with the alpha-thalassaemia-1 phenotype are homozygous for the alpha-thalassaemia-2 determinant. Restriction-endonuclease mapping shows that alpha-thalassaemia-2 results from the deletion of one of the linked pair of alpha-chain genes. Hence the genotypes of the alpha-thalassaemia heterozygotes and homozygotes in these families are -alpha/alpha alpha and -alpha/-alpha respectively. If these are the usual alpha-thalassaemia genotypes in Negroes, these findings explain the difference in clinical expression of the disorder between Orientals and Negroes--in particular, the absence of haemoglobin Bart's hydrops and the rarity of haemoglobin-H disease in Negroes.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated alpha-chain associated with an alpha + thalassaemia phenotype. The amount of alpha mRNA relative to beta mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of alpha + thalassaemia. In a patient with Hb CS-H disease there was greater alpha/beta mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the alpha mRNA in the patient's peripheral blood was derived from the alpha 1 (alpha A) gene. The data suggest that alpha CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3' non-coding region during translation.

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.

(1982). Sickle Cell-Hemoglobin D Iran: A Benign Sickle Cell Syndrome. Hemoglobin: Vol. 6, No. 1, pp. 57-59.

The recently identified θ-globin gene subfamily conists of the θ1-globin gene located downstream from the α1 -globin gene1–5, and several other members including at least one truncated, processed pseudogene ψθ2 (refs 1,6). Unlike the θ1 -globin genes of the rabbit3 and galago4, the structure of these genes in the orang-utan and baboon1,5 and their flanking regions show no apparent defects that would prevent their expression. Both θ1 -globin genes are split into three exons with the potential to code for a polypeptide of length 141 amino acids. Besides differing by 26% in replace-ment-site substitutions, the θ1 and α1 -globin genes of the orang-utan and baboon also differ in their promoter structures, in the use of TGA versus TAA as the termination codon, and in the use of AGTAAA versus AATAAA as the polyadenylation signal1,5. In contrast, the two θ1,-globin genes from primates only differ by 1.7% in the replacement-site substitutions5. Here we present the complete DNA sequence of a cloned θ1 -globin gene of humans, and show that it contains no apparent defects that would abolish its expression. Furthermore, by primer extension of single-stranded oligonucleotide probes, we show that the θ1 -globin gene of humans is transcribed in an erythroleukemia cell line K562. Three messenger RNA species were detected, with 5′-ends mapping to ∼70 base pairs (bp) downstream from a TAT A promoter sequence, at 8 bp downstream from a GGGCGG promoter sequence and at 40 bp upstream from the ATG inititrion codon, respectively. Haemin treatment of the K562 cells slightly enhances the level of the longest θ1-transcript. Our results provide strong evidence that the θ1 -globin gene of humans is transcriptionally active in cells of erythroid orign, and suggests the presence of a functional θ1-polypeptide in specific cells, possibly those of early erythroid tissue.

Ten patients with sickle cell (SS) disease from a Jamaican family were found to have unusually high levels of haemoglobin F for this population. Each of them has inherited one sickle cell gene on a chromosome characterized by an arrangement of restriction fragment length polymorphisms (haplotype) which is very rare in the Jamaican population. Genetic analysis of the family suggests that there is a determinant linked to the beta-globin gene cluster, characterized by this haplotype, which is responsible for increased haemoglobin F production in response to anaemia. Interestingly this particular haplotype appears to be common in patients with SS disease in eastern Saudi Arabia in whom a high level of haemoglobin F is the rule rather than the exception. Hence it is possible that this haplotype (++-++) acts as a genetic marker for elevated levels of haemoglobin F in sickle cell disease.