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Abstract
Over the past fifty 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 
and ß globin genes are normally regulated and documenting the effects of inherited mutations which 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 with increasing frequency. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression which, in turn, may eventually open up new avenues for improving the management of patients with common types of thalassemia.
and ß globin expression might eventually be manipulated to improve the management of patients with thalassemia. The Normal Structure and Expression of the Globin Gene Clusters
The synthesis of hemoglobin is controlled by two developmentally regulated multigene clusters: the -like globin cluster on chromosome 16 (5'-
-2-1–3'), and the ß-like globin cluster on chromosome 11 (5'-
-G
-A-ß-3') producing embryonic (22 Hb Portland, 22 Hb Gower I and 22 Hb Gower II) fetal (22, HbF) and adult (2ß2, HbA) hemoglobin (Figure 1
). Coordinated expression of the genes in each cluster at all stages of development is dependent on critical regulatory elements located upstream of the genes. In the ß cluster there are five such elements, collectively referred to as the locus control region (LCR) while in the cluster, there is a single known element referred to as HS-40. In normal individuals, the synthesis of and ß globin chains is finely balanced during terminal erythroid differentiation, giving rise to red cells of consistent size (MCV) and hemoglobin content (MCH). These two multigene clusters are among the most intensively studied of all mammalian genes and our current understanding of the details of their genetic and epigenetic regulation has been comprehensively reviewed elsewhere.1–3
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- and ß-Globin Chains
To date we know of more than 1000 naturally occurring inherited mutations that affect either the structure or synthesis of the - and ß-like globin chains.11,12 Thalassemia occurs when there is a deficiency in globin expression and ß thalassemia occurs when ß globin synthesis is downregulated. Many different mutant alleles have been selected to reach very high frequencies in tropical and subtropical regions of the world because heterozygotes are, to some extent, protected from the effects of falciparum malaria. Carriers of and ß thalassemia are clinically normal but the associated hypochromic microcytic blood picture requires careful diagnostic screening to differentiate it from other conditions with similar hematological pictures (e.g., iron deficiency and sideroblastic anemia). In areas where and ß thalassemia commonly occur, compound heterozygotes and homozygotes for these mutations may suffer from the clinically severe forms of thalassemia (HbH disease and Hb Bart’s hydrops fetalis) and ß thalassemia (ß thalassemia major and the less severe ß thalassemia intermedia).1,3 The pathophysiology and clinical syndromes of the thalassemias are shown in Figure 2.
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Thalassemia is the world’s most common monogenic disorder. In areas where thalassemia is prevalent, the carrier frequency of thalassemia varies from 1% (e.g., in Southern Spain) to 90% (e.g., the tribal populations of India). Similarly, the carrier frequency of ß thalassemia may vary from 1% (e.g., in Northern Italy) to 50%–70% (e.g., ßE in some regions of southeast Asia). Considerable variation occurs even within a single country. It has been estimated that worldwide there are 270 million carriers of mutant globin alleles which can potentially cause severe forms of thalassemia and hemoglobinopathy (including sickle cell disease), and every year some 300–400,000 severely affected infants are born.13 More than 95% of these affected births occur in Asia, India and the Middle East. However, over the past few decades there has been considerable mobility in the world’s populations so that now in Australasia, Europe and North America a significant proportion of recent immigrants originate from countries in which thalassemia commonly occurs. Therefore, the management of families from ethnic minorities, at risk of producing offspring with severe life-threatening forms of thalassemia, has become a new important aspect of hematologic practice. It has also been noted over the past 50 years that, as the world economy improves and the overall infant mortality rate falls, thalassemia is rapidly emerging as a major economic and health burden in developing countries since infants with thalassemia who would otherwise have died from infection and malnutrition now survive and require lifelong treatment. Therefore, worldwide, thalassemia is an enormous and ever-increasing hematologic problem in both developed and developing countries.14
Current Management and Outlook for Patients with Thalassemia and Their Families
Characterizing the molecular defects underlying and ß thalassemia has transformed the management of these conditions in many developed countries. Over the past 25 years, throughout Europe, Australasia and North America comprehensive control programs involving education, counselling and pre-natal diagnosis have succeeded in limiting the numbers of new births of affected individuals. One of the best-documented examples of this approach has been in Cyprus where thalassemia was first recognized in the 1940s following the eradication of malaria. With a population size of ~700,000 and a carrier frequency for ß thalassemia of 15%–17%, in the early 1970s it was estimated that to treat all severely affected children would require 78,000 units of blood per year, 40% of the population would be donors and the total cost to the health service would exceed the island’s health budget.15 With judicious application of a control program there are now fewer than 600 severely affected patients with thalassemia in Cyprus and only two or three new cases arising each year (Dr. M. Kleanothus, personal communication). A similar highly effective control program was also established by pioneering work in Sardinia.16
In First World countries the outlook for the relatively small numbers of patients with ß thalassemia major and intermedia has also dramatically improved over the last 25 years. Treatment protocols involving regular blood transfusion, which fully corrects the anemia, the careful use of splenectomy and the use of iron chelating drugs to remove the excess iron accumulated from transfusion has considerably increased the survival and quality of life for patients with ß thalassemia.17–20 In North America, for example, the median life span for such patients is approaching 40 years with over 90% of the adults in employment or working as full-time students (US Thalassemia Clinical Research Network Registry). Bone marrow transplantation is curative and, in the largest reported series, has been carried out in over 800 patients. The proportion of matched sibling donors is a limiting factor and the procedure is associated with an overall mortality of around 20%, the success depending largely on the clinical well being of the patient prior to transplantation.21
To date, the use of compounds that increase globin synthesis and the production of fetal Hb (see below) has been disappointing. Hydroxyurea increases the level of HbF and improves the clinical course of patients with S/ß thalassemia and homozygous sickle cell disease,22 although its precise mode of action is not clear. Hydroxyurea has not proved to be of similar benefit in patients with ß thalassemia. A few well-documented, albeit anecdotal, cases of ß thalassemia have responded well to other drugs (e.g., butyrate compounds) which also increase HbF production.3
These successful, although by no means perfect, approaches are readily available in developed countries, but these areas include less than 1% of the severely affected patients with ß thalassemia worldwide. The overwhelming majority of individuals with thalassemia die undiagnosed, untreated or at best only partially treated. Ultimately, with appropriate developments in screening, one might hope to reduce the number of affected births worldwide. In the meantime, a continuing challenge in thalassemia research is to find new ways to ameliorate the disease without such expense and without such a demanding infrastructure. Some clues to these approaches might be gained by investigating naturally occurring genetic interactions which significantly ameliorate the clinical phenotype of thalassemia.
Clinical Variability Explained in Terms of Pathophysiology and Modifiers of Disease Severity
From a world health point of view, ß thalassemia is the most important disorder of globin synthesis to consider, although some patients with HbH disease also have transfusion-dependent anemias.23 At present, approximately 200 different molecular defects which downregulate ß globin expression have been characterized.12 Homozygotes and compound heterozygotes for these alleles have variable clinical phenotypes ranging from severe transfusion-dependent anemia to mild asymptomatic hypochromic microcytic anemia. In all forms of ß thalassemia there is a reduction in the synthesis of ß globin chains which results in underproduction of HbA (Figure 2c
). However, the most deleterious effect on erythropoiesis occurs as a result of the precipitation of excess, unpaired globin chains in erythroblasts (causing dyserythropoiesis) and mature red cells (causing hemolysis).24 To a large extent, the clinical and hematologic outcome of the interaction of any two ß globin alleles depends on the degree to which ß globin expression is downregulated; this in turn can be predicted from precise knowledge of the molecular defects.
However, this oversimplifies the problem. It is well established, and commonly observed, that patients with identical ß globin genotypes may have radically different clinical phenotypes, ranging from severe transfusion-dependent ß thalassemia major to mild forms of thalassemia intermedia. This clinical heterogeneity has been the subject of intense study over the past 20 years. Although still not fully understood, two important genetic modifiers of phenotype have emerged, both of which influence the levels of free chains in erythroid cells (see above). Firstly, inheritance of cis- or trans-acting mutations which increase the ability of adult erythroblasts to synthesize globin chains, which can combine with excess chains to form HbF, can significantly ameliorate the clinical and hematologic features of ß thalassemia. Similarly, many studies have shown that co-inheritance of thalassemia can also reduce the pool of free chains and thereby influence the clinical severity of ß thalassemia.25–27 The primary importance of free chains in causing the phenotype of ß thalassemia has been underscored by many reports of patients who inherit just one abnormal ß globin allele (ßt/ß) but co-inherit five (/), six (/) or more globin genes. Rather than having the phenotype of ß thalassemia trait, such patients often have ß thalassemia intermedia.28,29
These key observations on natural variants which increase globin synthesis or decrease globin synthesis, and act as important modifiers of ß thalassemia, highlight two pathways that could be manipulated with therapeutic agents to redress the globin chain imbalance underlying ß thalassemia. Although not all patients with ß thalassemia would necessarily benefit, it seems likely that by both increasing and decreasing globin synthesis the spectrum of ß thalassemia could be shifted and the clinical outcome improved for substantial numbers of patients. To date nearly all scientific investigation and therapeutic intervention has been directed toward increasing globin synthesis and relatively little attention has been paid to understanding how one might reduce globin expression. Clearly this requires a detailed knowledge of how the globin genes are normally regulated.
Approaches to Understanding How the Globin Genes Are Normally Regulated During Hematopoiesis
Understanding in detail how the globin genes are regulated addresses this important clinical issue and, in a complementary way, contributes to a more general question in human molecular and cellular biology. In the post-genome era there is a major interest in understanding how, during development and differentiation, specific programs of expression emerge from the ~25,000 genes that are potentially available. While global approaches are being applied to this problem, a complementary approach is to understand how a single gene, such as globin, is activated or repressed as a pluripotent stem cell undergoes lineage specification and differentiation, for example during hematopoiesis. Our approach to understanding how the globin genes are normally regulated is set out below.
Defining the chromosomal segment containing all of the cis-acting sequences required for fully regulated expression of the globin genes
The human globin cluster lies very close (~150 kb) to the telomere of the short arm of chromosome 16, in a GC-rich, Alu dense, gene-dense segment of DNA.30 In fact the cluster is surrounded by widely expressed genes and its upstream regulatory elements (including HS-40) lie in the introns of an adjacent housekeeping gene (C16orf35; see Figure 3). To determine how much of the chromosome spanning the cluster might be required to fully regulate expression we have taken an evolutionary approach.31 It seemed likely that the minimal segment of the chromosome required for full regulation of globin expression should have been maintained throughout evolution. Such a segment might be defined by comparing multiple species and identifying the smallest region of conserved synteny. This approach has identified an 135–155 kb segment of DNA including the genes and all of its known regulatory elements (Figure 3). This region of conserved synteny has been maintained throughout over 500 million years of evolution.31
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Functional assays of the cis-elements
The potential role of each of the conserved non-coding cis-acting elements, on their own and in combination, has been tested when stably integrated into erythroid cell lines and transgenic mice.35–39 In the human cluster the only active elements in these experimental assays are the -like globin promoters and HS-40, a regulatory element lying 40 kb upstream of the globin genes. The individual roles of the other conserved elements remain to be determined. The most helpful guide to the potential role of these elements in vivo comes from the analysis of patients with thalassemia who have deletions of these elements (summarized in 40). Thalassemia most frequently results from deletion of the structural genes but deletions affecting the globin promoter have recently been described highlighting the critical importance of this region in vivo. A second group of rare deletions remove ~50–150 kb of the upstream region lying between the genes and the 16p telomere, where all the remaining conserved non-coding sequences are found (Figure 3). All of these deletions remove three of the conserved elements (HS-48, HS-40 and HS-33) but leave the genes themselves intact. Although small constructs containing various combinations of these conserved upstream elements linked to the -like genes direct tissue- and developmental stage-specific expression, none of them directs the high levels of expression seen from the endogenous globin genes in their natural chromosomal environment. However, more recent experiments from our own laboratory35 and others41 suggests that consistently higher levels of regulated expression may be seen in transgenic mice containing large fragments of DNA (~110–150 kb) including most if not all of the region of conserved synteny. In fact, such transgenes are able to fully rescue mice with no endogenous globin expression.42,43 This suggests that the critical cis-elements controlling globin gene expression may depend on their being within the correct chromatin environment to be fully functional, and this may only be recreated in the context of the large region of conserved synteny contained, for example, in a P1 artificial chromosome (PAC). The potential components of this chromatin environment are considered in the next section.
Chromosomal Environment of the and ß Globin Clusters and Epigenetic Changes During Hematopoiesis
Although gene expression may ultimately depend on the binding of transcriptional activators, which increase the rate of initiation or elongation of transcription, regulation of this process is greatly influenced by the way in which genes and their regulatory elements are packaged into chromatin. Chromatin packaging is complex and is associated with and/or influenced by a variety of interdependent nuclear processes including the sublocalization of the genes in the nucleus, the timing and replication in the cell cycle, histone modifications and DNA methylation. In general, silent chromatin co-localizes with heterochromatin and replicates late in the cell cycle; the associated chromatin is not acetylated and DNA is methylated. The opposite is generally true for active, accessible chromatin. Many hundreds of proteins have been shown to play a role in establishing and maintaining changes in chromatin accessibility, but two classes of proteins are particularly important. One group covalently modifies chromatin, for example by acetylating (e.g., p300/CBP) or de-acetylating (e.g., HDAC1) histones: many other functionally important chromatin modifications (methylation, phosphorylation, ATP-ribosylation, ubiquitination) have also been described.44
A second group consists of multiprotein complexes which act as molecular ‘motors’ which can remove or change the position of nucleosomes in chromatin thereby altering the accessibility of key cis-acting regulatory elements.45 Many of these modifications and chromatin-mediated effects can be stably inherited from one cell to its progeny and they are often referred to as epigenetic modifications.46
From studies spanning the last 20 years we now have an outline of the epigenetic changes that occur in the and ß globin clusters during erythropoiesis. As multipotent hemopoietic progenitors become committed to erythropoiesis both the and ß clusters bind erythroid-enriched transcription factors at their key cis-elements (e.g., -HS-40 and the ß-LCR) and this is associated with the development of histone modifications which occur with activation (e.g., acetylation) of chromatin and an increase in accessibility to DNA binding proteins, as judged by nuclease sensitivity. By contrast, in non-erythroid cells, the chromatin associated with the and ß clusters appears very different. Whereas the chromatin associated with the ß cluster has the characteristic features of silent chromatin, the cluster retains many of the features of active chromatin.35 Teleologically, this makes sense because the globin cluster is surrounded by housekeeping genes that must be expressed in both erythroid and non-erythroid cells.30
The important conclusion from these and other observations, accumulated over many years using indirect assays of chromatin structure and function (summarized in Table 1), is that despite their common evolutionary origin, during the last 500 million years the human and ß globin clusters have translocated to, and evolved in, quite different chromosomal environments. This in turn suggested that trans-acting mutations involving either transcription factors or proteins which influence the different epigenetic modifications associated with these two clusters might have quite different effects on and ß globin expression and thereby give rise to the phenotype of or ß thalassemia. It seemed possible that the identification of such mutations might point to new pathways through which to alter the relative expression of and ß globin in patients with thalassemia.
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Although the vast majority of mutations which cause thalassemia are linked to the and ß clusters, recently several rare conditions have been described in which and ß thalassemia have been caused by mutations in trans-acting factors. This was first illustrated in families with ß thalassemia rather than thalassemia. In one family, a mutation affecting the key erythroid transcription factor GATA-1 has been shown to cause thrombocytopenia and ß thalassemia.7 Subsequently, other families with similar mutations in GATA-1 causing ß thalassemia have been recognized (Vyas P et al, unpublished). It has been suggested that differences in the types of GATA-1 binding sites present in the and ß clusters may explain why this trans-acting mutation has different effects on expression of the two clusters.7
The second example of a trans-acting mutation causing ß thalassemia has been characterized in a group of 19 patients with a rare autosomal recessive disorder called trichothiodystrophy (TTD OMIM Catalog #601675) characterized by dry photosensitive skin, brittle hair, short stature and variable degrees of mental retardation. These individuals inherit mutations in one protein (XPD helicase) of the multicomponent, general transcription factor TFIIH.4,47 To date, all of these patients have the hematologic features of ß thalassemia trait. The simplest explanation for this is that the and ß promoters have different requirements for the activity of TFIIH in initiating transcription during erythropoiesis but the details of this are currently unknown.
There are several other families with ß thalassemia in whom no mutations have been detected in the genes or their regulatory elements indicating that other trans-acting mutations remain to be found (for example 48,49). In addition, it has been shown that there are genetic modifiers of globin expression which are unlinked to the ß globin cluster. These modifiers have been linked to chromosomes 6, 8 and X but the identity of these genes and their function are unknown.50–52
As for ß thalassemia, nearly all mutations causing thalassemia are cis-acting defects involving the genes, their promoters or the upstream regulatory elements. Nevertheless over the past 10 years two conditions (ATR-X syndrome and thalassemia myelodysplasia syndrome [ATMDS] syndrome) have been characterized in which mutations in a trans-acting factor called ATRX cause the phenotype of thalassemia. These are described below.
Mutations in a trans-Acting, Chromatin-Associated Protein Cause Thalassemia
Some years ago we reported the unusual association of thalassemia (HbH disease) with multiple developmental abnormalities53 in patients of North European origin. It is now clear that this association may occur as a result of two quite distinct mechanisms; one resulting from large deletions involving the telomeric region of chromosome 16 ( thalassemia with retardation on chromosome 16, ATR-16 syndrome OMIM catalog #141750); the other, caused by mutations in an unlinked X-encoded protein called ATRX ( thalassemia with retardation encoded on the X chromosome, ATR-X syndrome OMIM catalog #301040). The latter condition was immediately recognized as providing a new and potentially important insight into the trans-regulation of globin gene expression. Over 100 families with ATR-X syndrome have now been characterized and the core clinical features include severe psychomotor delay, a characteristic abnormal facial appearance, urogenital abnormalities and a variable degree of thalassemia.5,54
Although mutations in ATRX nearly always downregulate globin expression, at present its normal role in regulating gene expression is not clear and to address this we are at the stage of trying to pull together a series of somewhat disparate observations. In particular, we are documenting its pattern of expression, comparing it with similar proteins, establishing its sublocalization in the cell and identifying some of its protein partners, to develop an understanding of how ATRX normally works. ATRX is a large (280 kD) protein that is widely expressed throughout development. It contains two highly conserved subdomains. At the N-terminus is an extended PHD-like domain which is most closely related to similar domains found in the de novo methyltransferases (DNMT3a and b). At the C-terminus is a helicase/ATPase domain which provisionally classifies ATRX as a member of the SWI2/Snf2 family of molecular ‘motors’ which use the hydrolysis of ATP as a source of energy.5,55 These proteins are frequently present in multicomponent complexes that remodel chromatin and thereby influence the wide range of the epigenetic nuclear processes described above (e.g., DNA replication, DNA repair, recombination, transcription, DNA methylation).44,45 The recent isolation of endogenous ATRX has confirmed that it is an ATPase and, like other members of this family, has translocase activity: that is to say, in vitro, the protein can move along double-stranded DNA in the presence of ATP.56 The functional importance of the PHD-like and helicase domains has been demonstrated by the fact that nearly all inherited ATRX mutations fall within these two regions of the protein.54
Within cells, ATRX is localized in the nucleus and is concentrated in three distinct subcompartments: associated with heterochromatin,57 at rDNA arrays57 and in PML bodies.56 ATRX may also be present at more diffuse euchromatic loci, including the globin complex, and there appears to be some interchange of ATRX between these compartments. Proteins that have been shown to interact with ATRX (e.g., heterochromatin protein 157–59 and Daxx56) are consistent with these sublocalizations and may also shuttle between the same compartments. Although we have some insight into the function of heterochromatin (a repressive environment), PML bodies (containing proteins involved in regulation of gene expression, apoptosis and the cell cycle) and rDNA (the synthesis of rRNA for protein synthesis), the connection to ATRX has not yet been established. Perhaps the clearest example of a biological function for ATRX concerns its role in DNA methylation. It was of particular interest when it was shown that CpG methylation at heterochromatic loci is perturbed in patients with inherited mutations in ATRX suggesting that ATRX is involved in the establishment and/or maintenance of DNA methylation, an important epigenetic modifier.60
Despite these advances in our understanding of the localization and function of ATRX protein, it is frustrating that none of these insights has revealed how ATRX influences globin gene expression. The fact that ATRX mutations affect but apparently not ß globin expression may be informative. These two clusters are embedded in quite different chromosomal environments (see above), which supports the view that ATRX influences gene expression via one or more of the epigenetic features that distinguish these different regions. Current research priorities are to determine whether ATRX is detectable at the globin locus or its upstream regulatory sites and when during erythropoiesis ATRX mutations have their greatest effect.
On their own, these observations demonstrating a consistent but relatively modest effect of inherited ATRX mutations on globin expression are tantalizing but, to some extent, leave open the question of whether ATRX plays a central or peripheral role in the epigenetic regulation of globin gene expression. Showing that acquired mutations in ATRX virtually abolish globin expression (see below) has considerably strengthened and extended the potential importance of the ATRX multiprotein complex in regulating globin gene expression.
Thalassemia in the Context of Hematologic Malignancy
Although abnormal patterns of hemoglobin synthesis are nearly always inherited, occasionally individuals with previously normal hematology may develop aberrant hemoglobin synthesis as an acquired abnormality, usually within the context of hematologic malignancy.61 In 1960, two groups described a series of previously normal patients suffering from clonal hematopoietic disorders who developed an unusual acquired form of thalassemia during their illness.62,63 This syndrome was characterized by a marked hypochromic and microcytic anemia (Figure 4a; see Color Figures page 507) and the presence of HbH (ß4 tetramers) demonstrable by gel electrophoresis and supravital staining of peripheral red blood cells. (Figure 4b; see Color Figures page 507). Similar patients were soon found to have reduced /ß globin chain synthesis ratios, demonstrating unequivocally that thalassemia could occur as an acquired abnormality in the context of hematologic malignancy.64,65 In the light of these reports, a registry of patients with acquired thalassemia (www.imm.ox.ac.uk/groups/mrc_molhaem/home_pages/Higgs/index.html) was established in the early 1980s and currently includes 67 verified cases.
Acquired thalassemia is not limited to the geographical regions in which the inherited forms of thalassemia are common (Mediterranean region, Africa, Middle East, Asia). In fact, most patients have been of Northern European descent. A second, striking finding is that most patients with the acquired form of thalassemia reported to date have been male. In addition most have been elderly, with a median age at diagnosis of 68 years. As more cases have been evaluated, it has become clear that acquired thalassemia most commonly develops in the context of myelodysplastic syndrome (MDS) and this condition is now referred to as the " thalassemia myelodysplastic syndrome" (ATMDS, OMIM catalog #300448).
Most patients with MDS have an unexplained macrocytic or unremarkable normocytic anemia. Based on hematologic data from the registry, we can now say that ATMDS will be most frequently found in patients with MDS who have the typical abnormal red cell morphology (Figure 4a; see Color Figures page 507), an MCV of less than 80 fL and an MCH of less than 26 pg (Figure 5). To confirm the diagnosis it is still essential to perform an independent assessment for HbH inclusions when a diagnosis of ATMDS is suspected on the criteria set out above. Since many clinical facilities no longer offer supravital staining, our laboratory provides this service free of charge for suspected ATMDS patients, if sent 1 mL of fresh blood by courier.
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globin expression is downregulated in ATMDS (e.g., transcription, mRNA processing translation). The /ß globin mRNA ratio has only been studied in a few ATMDS cases and was reduced to < 0.5 in all patients analyzed.64,66 The /ß globin chain synthesis ratio is strikingly abnormal (often < 0.2), which is less than one would expect to see in patients with only a single functional globin gene (--/-), suggesting that in many ATMDS cases, all four globin genes are downregulated. The globin gene cluster itself has been carefully analyzed by Southern blot hybridization and direct sequencing and with one exception was found to be entirely normal, supporting the hypothesis that ATMDS results from a trans-acting mutation. A major clue toward identifying the molecular defect in ATMDS came from a cDNA microarray study of peripheral blood neutrophil RNA from a man newly diagnosed with ATMDS compared with pooled granulocyte RNA from several normal individuals. This demonstrated that the ATRX gene at Xq13.3 was one of the genes with the lowest expression in the ATMDS patient compared to controls, a result that was confirmed by RT-PCR. An ATRX point mutation (G>A) was then found in the canonical splice donor site of intron 1 in this patient, which likely resulted in nonsense-mediated decay of the truncated ATRX transcript.9
In light of this encouraging finding, archival material including unfractionated blood and marrow from ATMDS registry patients was screened by denaturing high-performance liquid chromatography, which is sensitive to low-level mosaicism, and by sequencing of the 10 kb ATRX mRNA. This revealed 11 additional patients with point mutations and/or splicing abnormalities in ATRX. The mutations clustered in and near the PHD-like and helicase domains (Figure 6) which are commonly mutated in the inherited ATR-X syndrome.10
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Globin Expression. What Does It Tell Us About the Molecular Basis of MDS?
These findings have demonstrated that both inherited and acquired mutations in ATRX cause thalassemia, suggesting that ATRX normally plays a central role in the regulation of globin gene expression. The widespread developmental abnormalities in ATR-X syndrome also suggest that ATRX plays a more general role in gene regulation. However, at present the mechanism by which ATRX controls gene expression is not clear. Nevertheless, many different lines of evidence suggest that ATRX is a chromatin associated protein which may act as a transcriptional co-factor and/or influence the epigenetic control of gene expression. The discovery of ATRX mutations in ATMDS has also highlighted several other unanswered questions. Solutions to these puzzles may provide important insights into how ATRX normally regulates gene expression and may also uncover some common molecular defects underlying MDS that have more general importance in the area of molecular hematology.
It is curious that the hematologic phenotype in ATMDS patients is, in general, much more severe than that seen in boys who have congenital ATR-X syndrome.10 For example, a boy with a germline 576G>C; L192F ATRX mutation had 0.1% HbH-containing erythrocytes, while an ATMDS patient who had acquired an identical mutation (restricted to myeloid cells) had 50% HbH cells. As described above, patients with ATMDS usually have very low /ß globin synthesis ratios (< 0.2 in 52% of patients) and large amounts of HbH (median 30%) compared to boys with inherited ATR-X syndrome, who generally have only mildly reduced /ß synthesis ratios with low and sometimes undetectable amounts of HbH. Since the spectrum of ATRX mutations is similar in these congenital and acquired forms of HbH disease (Figure 6), this suggests that the different hematologic effects of ATRX mutations in erythroid cells depends on either when the mutation occurs in development or, more likely, the cellular context in which the mutation occurs.
Could there be a relatively common pattern of genetic or epigenetic changes in the erythroblasts of patients with MDS that drastically enhances the deleterious effects of ATRX mutations on globin expression? The myeloid cells of patients with MDS often have many different genetic mutations, the full repertoire of which is only just beginning to be defined, and recent reports also demonstrate widespread MDS-associated epigenetic abnormalities reflected in the patterns of DNA methylation and histone acetylation.67–69 Current, incomplete observations suggest that globin expression per se is not altered in patients with MDS. Therefore, it seems more likely that, in the context of MDS, there are genetic or epigenetic mutations that directly or indirectly exacerbate the deleterious effects of an ATRX mutation. Given the presumed rarity of ATRX mutations, these interacting mutations might occur quite frequently in all types of MDS. Our knowledge of the sublocalization of ATRX and its interacting protein partners (Figure 7; see Color Figures page 508) provides several candidates for potential modifying genetic and epigenetic factors that may be commonly altered in MDS, and we are currently systematically screening for mutations in these partners.
Conclusions
Despite many advances over the past fifty years, the thalassemia syndromes still represent a major worldwide health problem and are still amongst the most common monogenic diseases affecting man. Comprehensive characterization of over ~200 molecular defects causing ß thalassemia and ~100 causing thalassemia have enabled many countries in Australasia, Europe and North America to establish comprehensive screening programs to prevent new cases arising. Although these programs address the recent immigration of individuals from areas where thalassemia commonly occurs, they reach less than 1% of the total population at risk. For new cases of severe thalassemia that arise in developed countries current therapy is effective but expensive and burdensome both for the patient and the health care system. New approaches to the management of these common diseases in both affluent and developing nations are urgently required.
One approach is to learn from and mimic the naturally occurring genetic interactions which significantly ameliorate the clinical phenotype of ß thalassemia. The best examples of this are those conditions which increase the expression of fetal () globin chains or decrease the synthesis of globin chains. Our work has concentrated on the latter approach. By understanding in detail how the globin genes are normally regulated during hematopoiesis and again from the analysis of inherited and acquired natural mutants, we have gained many new insights. Of particular importance, it appears that the epigenetic program regulating globin expression is quite different from that associated with the ß globin cluster. This work points to new pathways which may be targets for therapeutic agents that specifically downregulate but not ß globin expression.
Footnotes
* MD, DSc(Med); MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK 
References
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-thalassaemia in -globin knockout mice with a 191 kb transgene containing the human -globin locus. Transgenic Research. 2004;2120:1–9.[CrossRef]
and ß globin gene expression. Weatherall Institute of Molecular Medicine. Oxford: Oxford University; 2002.
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