Prof Doug Higgs FRS

Research Area: Cell and Molecular Biology
Technology Exchange: Bioinformatics, Chromosome mapping, Computational biology and In situ hybridisation
Keywords: Genomics, gene regulation, genetics, epigenetics
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Our laboratory is interested in the general question of how mammalian genes are switched on and off during lineage commitment and differentiation. We study genes (e.g. globin) in detail and also study gene expression using genome wide analyses. We study all aspects of gene expression including the key cis-regulatory elements (enhancers, promoters and insulators), the transcription factors and co-factors that bind them, the epigenetic modifications of chromatin and DNA and the role of associated phenomena such as chromosome conformation and nuclear sub-compartmentalisation using imaging techniques. These studies are performed both in cell systems and in model organisms as well as in human patients with various inherited and acquired genetic and epigenetic abnormalities. The translational goal of this work is to develop new ways to modify gene expression during blood formation with the aim of manipulating gene expression and ameliorating the clinical phenotypes of patients with a variety of blood disorders.

Name Department Institution Country
Prof Richard Gibbons FMedSci FRCP Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Sir David Weatherall KBE FRS FMedSci Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Veronica J Buckle Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Jim R Hughes Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Graham B, Marcais A, Dharmalingam G, Carroll T, Kanellopoulou C, Graumann J, Nesterova TB, Bermange A, Brazauskas P, Xella B et al. 2016. MicroRNAs of the miR-290-295 Family Maintain Bivalency in Mouse Embryonic Stem Cells. Stem Cell Reports, 6 (5), pp. 635-642. | Show Abstract | Read more

Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.

Perkins A, Xu X, Higgs DR, Patrinos GP, Arnaud L, Bieker JJ, Philipsen S, KLF1 Consensus Workgroup. 2016. Krüppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants. Blood, 127 (15), pp. 1856-1862. | Show Abstract | Read more

Until recently our approach to analyzing human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, in thalassemia, the globin loci are analyzed. Sequencing has become increasingly accessible, and thus a larger panel of genes can be analyzed and whole exome and/or whole genome sequencing can be used when no variants are found in the candidate genes. By using such approaches in patients with unexplained anemias, we have discovered that a broad range of hitherto unrelated human red cell disorders are caused by variants in KLF1, a master regulator of erythropoiesis, which were previously considered to be extremely rare causes of human genetic disease.

Masuda T, Wang X, Maeda M, Canver MC, Sher F, Funnell AP, Fisher C, Suciu M, Martyn GE, Norton LJ et al. 2016. Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin. Science, 351 (6270), pp. 285-289. | Show Abstract | Read more

Genes encoding human β-type globin undergo a developmental switch from embryonic to fetal to adult-type expression. Mutations in the adult form cause inherited hemoglobinopathies or globin disorders, including sickle cell disease and thalassemia. Some experimental results have suggested that these diseases could be treated by induction of fetal-type hemoglobin (HbF). However, the mechanisms that repress HbF in adults remain unclear. We found that the LRF/ZBTB7A transcription factor occupies fetal γ-globin genes and maintains the nucleosome density necessary for γ-globin gene silencing in adults, and that LRF confers its repressive activity through a NuRD repressor complex independent of the fetal globin repressor BCL11A. Our study may provide additional opportunities for therapeutic targeting in the treatment of hemoglobinopathies.

Mettananda S, Gibbons RJ, Higgs DR. 2016. Understanding α-globin gene regulation and implications for the treatment of β-thalassemia. Ann N Y Acad Sci, 1368 (1), pp. 16-24. | Show Abstract | Read more

Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality.

Wills QF, Higgs DR, Mead AJ. 2015. Studying epigenomics in single cells: what is feasible and what can we learn? Epigenomics, 7 (8), pp. 1231-1234. | Read more

Davies JO, Telenius JM, McGowan SJ, Roberts NA, Taylor S, Higgs DR, Hughes JR. 2016. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat Methods, 13 (1), pp. 74-80. | Show Abstract | Read more

Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.

Blobel GA, Bodine D, Brand M, Crispino J, de Bruijn MF, Nathan D, Papayannopoulou T, Porcher C, Strouboulis J, Zon L et al. 2015. An international effort to cure a global health problem: A report on the 19th Hemoglobin Switching Conference. Exp Hematol, 43 (10), pp. 821-837. | Show Abstract | Read more

Every 2 years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action, and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19th Hemoglobin Switching Conference, held in September 2014 at St. John's College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental, and genomic advances focused on these diseases. The conference comprised about 60 short talks over 3 days by leading investigators in the field. This meeting report describes the highlights of the conference.

Mettananda S, Gibbons RJ, Higgs DR. 2015. α-Globin as a molecular target in the treatment of β-thalassemia. Blood, 125 (24), pp. 3694-3701. | Show Abstract | Read more

The thalassemias, together with sickle cell anemia and its variants, are the world's most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia.

Clynes D, Jelinska C, Xella B, Ayyub H, Scott C, Mitson M, Taylor S, Higgs DR, Gibbons RJ. 2015. Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nat Commun, 6 pp. 7538. | Show Abstract | Read more

Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers.

Voon HP, Hughes JR, Rode C, De La Rosa-Velázquez IA, Jenuwein T, Feil R, Higgs DR, Gibbons RJ. 2015. ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes. Cell Rep, 11 (3), pp. 405-418. | Show Abstract | Read more

Histone H3.3 is a replication-independent histone variant, which replaces histones that are turned over throughout the entire cell cycle. H3.3 deposition at euchromatin is dependent on HIRA, whereas ATRX/Daxx deposits H3.3 at pericentric heterochromatin and telomeres. The role of H3.3 at heterochromatic regions is unknown, but mutations in the ATRX/Daxx/H3.3 pathway are linked to aberrant telomere lengthening in certain cancers. In this study, we show that ATRX-dependent deposition of H3.3 is not limited to pericentric heterochromatin and telomeres but also occurs at heterochromatic sites throughout the genome. Notably, ATRX/H3.3 specifically localizes to silenced imprinted alleles in mouse ESCs. ATRX KO cells failed to deposit H3.3 at these sites, leading to loss of the H3K9me3 heterochromatin modification, loss of repression, and aberrant allelic expression. We propose a model whereby ATRX-dependent deposition of H3.3 into heterochromatin is normally required to maintain the memory of silencing at imprinted loci.

Viprakasit V, Ekwattanakit S, Riolueang S, Chalaow N, Fisher C, Lower K, Kanno H, Tachavanich K, Bejrachandra S, Saipin J et al. 2014. Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression. Blood, 123 (10), pp. 1586-1595. | Show Abstract | Read more

In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.

Bassett AR, Akhtar A, Barlow DP, Bird AP, Brockdorff N, Duboule D, Ephrussi A, Ferguson-Smith AC, Gingeras TR, Haerty W et al. 2014. Considerations when investigating lncRNA function in vivo. Elife, 3 (August2014), pp. e03058. | Show Abstract | Read more

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

Hughes JR, Roberts N, McGowan S, Hay D, Giannoulatou E, Lynch M, De Gobbi M, Taylor S, Gibbons R, Higgs DR. 2014. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat Genet, 46 (2), pp. 205-212. | Show Abstract | Read more

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.

Funnell AP, Vernimmen D, Lim WF, Mak KS, Wienert B, Martyn GE, Artuz CM, Burdach J, Quinlan KG, Higgs DR et al. 2014. Differential regulation of the α-globin locus by Krüppel-like Factor 3 in erythroid and non-erythroid cells. BMC Mol Biol, 15 (1), pp. 8. | Show Abstract | Read more

BACKGROUND: Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. RESULTS: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3-/- mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3-/- erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. CONCLUSIONS: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.

Clynes D, Jelinska C, Xella B, Ayyub H, Taylor S, Mitson M, Bachrati CZ, Higgs DR, Gibbons RJ. 2014. ATRX dysfunction induces replication defects in primary mouse cells. PLoS One, 9 (3), pp. e92915. | Show Abstract | Read more

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

Graham BJ, Hay D, Hughes J, Higgs D. 2014. The worm has turned: unexpected similarities between the transcription of enhancers and promoters in the worm and mammalian genomes. Bioessays, 36 (2), pp. 157-162. | Show Abstract | Read more

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.

Marques AC, Hughes J, Graham B, Kowalczyk MS, Higgs DR, Ponting CP. 2013. Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs. Genome Biol, 14 (11), pp. R131. | Show Abstract | Read more

BACKGROUND: Mammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers. RESULTS: We integrated high-resolution maps of transcriptional initiation and transcription to annotate a conservative set of intergenic lncRNAs expressed in mouse erythroblasts. We subclassified intergenic lncRNAs according to chromatin status at transcriptional initiation regions, defined by relative levels of histone H3K4 mono- and trimethylation. These transcripts are almost evenly divided between those arising from enhancer-associated (elncRNA) or promoter-associated (plncRNA) elements. These two classes of 5' capped and polyadenylated RNA transcripts are indistinguishable with regard to their length, number of exons or transcriptional orientation relative to their closest neighboring gene. Nevertheless, elncRNAs are more tissue-restricted, less highly expressed and less well conserved during evolution. Of considerable interest, we found that expression of elncRNAs, but not plncRNAs, is associated with enhanced expression of neighboring protein-coding genes during erythropoiesis. CONCLUSIONS: We have determined globally the sites of initiation of intergenic lncRNAs in erythroid cells, allowing us to distinguish two similarly abundant classes of transcripts. Different correlations between the levels of elncRNAs, plncRNAs and expression of neighboring genes suggest that functional lncRNAs from the two classes may play contrasting roles in regulating the transcript abundance of local or distal loci.

Clynes D, Higgs DR, Gibbons RJ. 2013. The chromatin remodeller ATRX: a repeat offender in human disease. Trends Biochem Sci, 38 (9), pp. 461-466. | Show Abstract | Read more

The regulation of chromatin structure is of paramount importance for a variety of fundamental nuclear processes, including gene expression, DNA repair, replication, and recombination. The ATP-dependent chromatin-remodelling factor ATRX (α thalassaemia/mental retardation X-linked) has emerged as a key player in each of these processes. Exciting recent developments suggest that ATRX plays a variety of key roles at tandem repeat sequences within the genome, including the deposition of a histone variant, prevention of replication fork stalling, and the suppression of a homologous recombination-based pathway of telomere maintenance. Here, we provide a mechanistic overview of the role of ATRX in each of these processes, and propose how they may be connected to give rise to seemingly disparate human diseases.

Hosseini M, Goodstadt L, Hughes JR, Kowalczyk MS, de Gobbi M, Otto GW, Copley RR, Mott R, Higgs DR, Flint J. 2013. Causes and consequences of chromatin variation between inbred mice. PLoS Genet, 9 (6), pp. e1003570. | Show Abstract | Read more

Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS). Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10%) of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.

Babbs C, Roberts NA, Sanchez-Pulido L, McGowan SJ, Ahmed MR, Brown JM, Sabry MA, WGS500 Consortium, Bentley DR, McVean GA et al. 2013. Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I. Haematologica, 98 (9), pp. 1383-1387. | Show Abstract | Read more

The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.

Lower KM, De Gobbi M, Hughes JR, Derry CJ, Ayyub H, Sloane-Stanley JA, Vernimmen D, Garrick D, Gibbons RJ, Higgs DR. 2013. Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression. Hum Mutat, 34 (8), pp. 1140-1148. | Show Abstract | Read more

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.

Hughes JR, Lower KM, Dunham I, Taylor S, De Gobbi M, Sloane-Stanley JA, McGowan S, Ragoussis J, Vernimmen D, Gibbons RJ, Higgs DR. 2013. High-resolution analysis of cis-acting regulatory networks at the α-globin locus Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 368 (1620), pp. 20120361. | Show Abstract | Read more

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.

Hughes JR, Lower KM, Dunham I, Taylor S, De Gobbi M, Sloane-Stanley JA, McGowan S, Ragoussis J, Vernimmen D, Gibbons RJ, Higgs DR. 2013. High-resolution analysis of cis-acting regulatory networks at the α-globin locus. Philos Trans R Soc Lond B Biol Sci, 368 (1620), pp. 20120361. | Show Abstract | Read more

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.

Higgs DR. 2013. The molecular basis of α-thalassemia. Cold Spring Harb Perspect Med, 3 (1), pp. a011718. | Show Abstract | Read more

The globin gene disorders including the thalassemias are among the most common human genetic diseases with more than 300,000 severely affected individuals born throughout the world every year. Because of the easy accessibility of purified, highly specialized, mature erythroid cells from peripheral blood, the hemoglobinopathies were among the first tractable human molecular diseases. From the 1970s onward, the analysis of the large repertoire of mutations underlying these conditions has elucidated many of the principles by which mutations occur and cause human genetic diseases. This work will summarize our current knowledge of the α-thalassemias, illustrating how detailed analysis of this group of diseases has contributed to our understanding of the general molecular mechanisms underlying many orphan and common diseases.

Kowalczyk MS, Hughes JR, Babbs C, Sanchez-Pulido L, Szumska D, Sharpe JA, Sloane-Stanley JA, Morriss-Kay GM, Smoot LB, Roberts AE et al. 2012. Nprl3 is required for normal development of the cardiovascular system. Mamm Genome, 23 (7-8), pp. 404-415. | Show Abstract | Read more

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.

Kowalczyk MS, Higgs DR, Gingeras TR. 2012. Molecular biology: RNA discrimination. Nature, 482 (7385), pp. 310-311. | Read more

Kowalczyk MS, Hughes JR, Garrick D, Lynch MD, Sharpe JA, Sloane-Stanley JA, McGowan SJ, De Gobbi M, Hosseini M, Vernimmen D et al. 2012. Intragenic enhancers act as alternative promoters. Mol Cell, 45 (4), pp. 447-458. | Show Abstract | Read more

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.

Lynch MD, Smith AJ, De Gobbi M, Flenley M, Hughes JR, Vernimmen D, Ayyub H, Sharpe JA, Sloane-Stanley JA, Sutherland L et al. 2012. An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment. EMBO J, 31 (2), pp. 317-329. | Show Abstract | Read more

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.

Higgs DR, Engel JD, Stamatoyannopoulos G. 2012. Thalassaemia. Lancet, 379 (9813), pp. 373-383. | Show Abstract | Read more

Thalassaemia is one of the most common genetic diseases worldwide, with at least 60,000 severely affected individuals born every year. Individuals originating from tropical and subtropical regions are most at risk. Disorders of haemoglobin synthesis (thalassaemia) and structure (eg, sickle-cell disease) were among the first molecular diseases to be identified, and have been investigated and characterised in detail over the past 40 years. Nevertheless, treatment of thalassaemia is still largely dependent on supportive care with blood transfusion and iron chelation. Since 1978, scientists and clinicians in this specialty have met regularly in an international effort to improve the management of thalassaemia, with the aim of increasing the expression of unaffected fetal genes to improve the deficiency in adult β-globin synthesis. In this Seminar we discuss important advances in the understanding of the molecular and cellular basis of normal and abnormal expression of globin genes. We will summarise new approaches to the development of tailored pharmacological agents to alter regulation of globin genes, the first trial of gene therapy for thalassaemia, and future prospects of cell therapy.

Vernimmen D, Lynch MD, De Gobbi M, Garrick D, Sharpe JA, Sloane-Stanley JA, Smith AJ, Higgs DR. 2011. Polycomb eviction as a new distant enhancer function. Genes Dev, 25 (15), pp. 1583-1588. | Show Abstract | Read more

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.

Renella R, Roberts NA, Brown JM, De Gobbi M, Bird LE, Hassanali T, Sharpe JA, Sloane-Stanley J, Ferguson DJP, Cordell J et al. 2011. Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1 alpha localization in erythroblasts BLOOD, 117 (25), pp. 6928-6938. | Show Abstract | Read more

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material byWestern blot.Adetailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genomewide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1gt/gt homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis. © 2011 by The American Society of Hematology.

Mitson M, Kelley LA, Sternberg MJ, Higgs DR, Gibbons RJ. 2011. Functional significance of mutations in the Snf2 domain of ATRX. Hum Mol Genet, 20 (13), pp. 2603-2610. | Show Abstract | Read more

ATRX is a member of the Snf2 family of chromatin-remodelling proteins and is mutated in an X-linked mental retardation syndrome associated with alpha-thalassaemia (ATR-X syndrome). We have carried out an analysis of 21 disease-causing mutations within the Snf2 domain of ATRX by quantifying the expression of the ATRX protein and placing all missense mutations in their structural context by homology modelling. While demonstrating the importance of protein dosage to the development of ATR-X syndrome, we also identified three mutations which primarily affect function rather than protein structure. We show that all three of these mutant proteins are defective in translocating along DNA while one mutant, uniquely for a human disease-causing mutation, partially uncouples adenosine triphosphate (ATP) hydrolysis from DNA binding. Our results highlight important mechanistic aspects in the development of ATR-X syndrome and identify crucial functional residues within the Snf2 domain of ATRX. These findings are important for furthering our understanding of how ATP hydrolysis is harnessed as useful work in chromatin remodelling proteins and the wider family of nucleic acid translocating motors.

Giardine B, Borg J, Higgs DR, Peterson KR, Philipsen S, Maglott D, Singleton BK, Anstee DJ, Basak AN, Clark B et al. 2011. Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach. Nat Genet, 43 (4), pp. 295-301. | Show Abstract | Read more

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Renella R, Roberts NA, Brown JM, De Gobbi M, Bird LE, Hassanali T, Sharpe JA, Sloane-Stanley J, Ferguson DJ, Cordell J et al. 2011. Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts. Blood, 117 (25), pp. 6928-6938. | Show Abstract | Read more

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.

Merryweather-Clarke AT, Atzberger A, Soneji S, Gray N, Clark K, Waugh C, McGowan SJ, Taylor S, Nandi AK, Wood WG et al. 2011. Global gene expression analysis of human erythroid progenitors. Blood, 117 (13), pp. e96-108. | Show Abstract | Read more

Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. [corrected].

Eustermann S, Yang JC, Law MJ, Amos R, Chapman LM, Jelinska C, Garrick D, Clynes D, Gibbons RJ, Rhodes D et al. 2011. Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin. Nat Struct Mol Biol, 18 (7), pp. 777-782. | Show Abstract | Read more

Accurate read-out of chromatin modifications is essential for eukaryotic life. Mutations in the gene encoding X-linked ATRX protein cause a mental-retardation syndrome, whereas wild-type ATRX protein targets pericentric and telomeric heterochromatin for deposition of the histone variant H3.3 by means of a largely unknown mechanism. Here we show that the ADD domain of ATRX, in which most syndrome-causing mutations occur, engages the N-terminal tail of histone H3 through two rigidly oriented binding pockets, one for unmodified Lys4 and the other for di- or trimethylated Lys9. In vivo experiments show this combinatorial readout is required for ATRX localization, with recruitment enhanced by a third interaction through heterochromatin protein-1 (HP1) that also recognizes trimethylated Lys9. The cooperation of ATRX ADD domain and HP1 in chromatin recruitment results in a tripartite interaction that may span neighboring nucleosomes and illustrates how the 'histone-code' is interpreted by a combination of multivalent effector-chromatin interactions.

De Gobbi M, Garrick D, Lynch M, Vernimmen D, Hughes JR, Goardon N, Luc S, Lower KM, Sloane-Stanley JA, Pina C et al. 2011. Generation of bivalent chromatin domains during cell fate decisions. Epigenetics Chromatin, 4 (1), pp. 9. | Show Abstract | Read more

BACKGROUND: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. RESULTS: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. CONCLUSIONS: While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.

Serjeant GR, Serjeant BE, Fraser RA, Hambleton IR, Higgs DR, Kulozik AE, Donaldson A. 2011. Hb S-β-thalassemia: molecular, hematological and clinical comparisons. Hemoglobin, 35 (1), pp. 1-12. | Show Abstract | Read more

Clinical and hematological features are presented for 261 patients with identified β-thalassemia (β-thal) mutations. Mutations causing Hb S [β6(A3)Glu→Val]-β(0)-thal were IVS-II-849 (A>G) in 44%, frameshift codon (FSC) 6 (-A) in 14%, Hb Monroe [β30(B12)Arg→Thr] in 14%, and IVS-II-1 (G>A) in 10%. Mutations causing Hb S-β(+)-thal with 14-25% Hb A (type III) were -29 (A>G) mutation in 60%, -88 (C>T) in 22% and the polyadenylation signal site (polyA) (T>C) mutation in 14%, and in Hb S-β(+)-thal with 1-7% Hb A (type I), all had the IVS-I-5 (G>C) mutation. Hematologically, only minor differences occurred between the four Hb S-β(0)-thal mutations, but among the three mutations causing Hb S-β(+)-thal type III, levels of Hb A(2), Hb F, hemoglobin (Hb), MCV and MCH were highest in the -88 and lowest in the polyA mutations. Clinically, Hb S-β(0)-thal and Hb S-β(+)-thal type I were generally severe, and Hb S-β(+)-thal type III disease with the -88 mutation was milder than that caused by the polyA mutation.

Cited:

69

Scopus

Giardine B, Borg J, Higgs DR, Peterson KR, Philipsen S, Maglott D, Singleton BK, Anstee DJ, Basak AN, Clark B et al. 2011. Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach Nature Genetics, 43 (4), pp. 295-302. | Show Abstract | Read more

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases. © 2011 Nature America, Inc. All rights reserved.

Higgs DR, Engel JD, Stamatoyannopoulos G. 2011. Thalassaemia The Lancet,

Gibbons RJ, Higgs DR. 2010. ATRX: taming tandem repeats. Cell Cycle, 9 (23), pp. 4605-4606. | Read more

Higgs DR, Gibbons RJ. 2010. The molecular basis of α-thalassemia: a model for understanding human molecular genetics. Hematol Oncol Clin North Am, 24 (6), pp. 1033-1054. | Show Abstract | Read more

Down-regulation of α-globin synthesis causes α-thalassemia with underproduction of fetal (HbF, α(2)γ(2)) and adult (HbA, α(2)β(2)) hemoglobin. This article focuses on the human α-globin cluster, which has been characterized in great depth over the past 30 years. In particular the authors describe how the α genes are normally switched on during erythropoiesis and switched off as hematopoietic stem cells commit to nonerythroid lineages. In addition, the principles by which α-globin expression may be perturbed by natural mutations that cause α-thalassemia are reviewed.

Law MJ, Lower KM, Voon HP, Hughes JR, Garrick D, Viprakasit V, Mitson M, De Gobbi M, Marra M, Morris A et al. 2010. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell, 143 (3), pp. 367-378. | Show Abstract | Read more

ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.

Orkin SH, Higgs DR. 2010. Medicine. Sickle cell disease at 100 years. Science, 329 (5989), pp. 291-292. | Read more

Harteveld CL, Higgs DR. 2010. Alpha-thalassaemia. Orphanet J Rare Dis, 5 (1), pp. 13. | Show Abstract | Read more

Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia.It is probably the most common monogenic gene disorder in the world and is especially frequent in Mediterranean countries, South-East Asia, Africa, the Middle East and in the Indian subcontinent. During the last few decades the incidence of alpha thalassaemia in North-European countries and Northern America has increased because of demographic changes. Compound heterozygotes and some homozygotes have a moderate to severe form of alpha thalassaemia called HbH disease. Hb Bart's hydrops foetalis is a lethal form in which no alpha-globin is synthesized. Alpha thalassaemia most frequently results from deletion of one or both alpha genes from the chromosome and can be classified according to its genotype/phenotype correlation. The normal complement of four functional alpha-globin genes may be decreased by 1, 2, 3 or all 4 copies of the genes, explaining the clinical variation and increasing severity of the disease. All affected individuals have a variable degree of anaemia (low Hb), reduced mean corpuscular haemoglobin (MCH/pg), reduced mean corpuscular volume (MCV/fl) and a normal/slightly reduced level of HbA2. Molecular analysis is usually required to confirm the haematological observations (especially in silent alpha-thalassaemia and alpha-thalassaemia trait). The predominant features in HbH disease are anaemia with variable amounts of HbH (0.8-40%). The type of mutation influences the clinical severity of HbH disease. The distinguishing features of the haemoglobin Bart's hydrops foetalis syndrome are the presence of Hb Bart's and the total absence of HbF. The mode of transmission of alpha thalassaemia is autosomal recessive. Genetic counselling is offered to couples at risk for HbH disease or haemoglobin Bart's Hydrops Foetalis Syndrome. Carriers of alpha+- or alpha0-thalassaemia alleles generally do not need treatment. HbH patients may require intermittent transfusion therapy especially during intercurrent illness. Most pregnancies in which the foetus is known to have the haemoglobin Bart's hydrops foetalis syndrome are terminated due to the increased risk of both maternal and foetal morbidity.

Goldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Dewell S, Law M, Guo X, Li X et al. 2010. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell, 140 (5), pp. 678-691. | Show Abstract | Read more

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Lower KM, Hughes JR, De Gobbi M, Henderson S, Viprakasit V, Fisher C, Goriely A, Ayyub H, Sloane-Stanley J, Vernimmen D et al. 2009. Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition. Proc Natl Acad Sci U S A, 106 (51), pp. 21771-21776. | Show Abstract | Read more

It is well established that all of the cis-acting sequences required for fully regulated human alpha-globin expression are contained within a region of approximately 120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the alpha-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the alpha-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of alpha-globin genes, affects the pattern of NME4 expression by altering the competition for the shared alpha-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.

Garrick D, De Gobbi M, Gibbons R, Higgs DR. 2009. CTCF, cohesin and higher-order chromatin structure. Epigenomics, 1 (2), pp. 232.

Garrick D, De Gobbi M, Gibbons R, Higgs DR. 2009. Polycomb response elements in vertebrates. Epigenomics, 1 (2), pp. 231. | Read more

Serjeant GR, Serjeant BE, Mason KP, Hambleton IR, Fisher C, Higgs DR. 2009. The changing face of homozygous sickle cell disease: 102 patients over 60 years. Int J Lab Hematol, 31 (6), pp. 585-596. | Show Abstract | Read more

Earlier reports on homozygous sickle cell (SS) disease have been biased by severely affected cases. The Jamaican clinic which seeks to avoid such bias has 102 patients surviving beyond 60 years. The objective of this study was to examine the features of elderly cases and assess factors determining survival and the behaviour of this disease with advancing age. A retrospective review of all cases and prospective assessment in survivors was conducted at The Sickle Cell Clinic at the University of the West Indies, Kingston, Jamaica previously operated by the MRC Laboratories. All patients with SS disease born prior to December 31, 1943 who would, by January 2004, have passed their 60th birthday were traced and their current status ascertained. The molecular and clinical features were assessed and observations on the clinical behaviour of the disease and of haematology and biochemistry are presented. Of the 102 patients, 58 had died, four had emigrated and 40 were alive, resident in Jamaica and aged 60-87 years. Survival was associated with female gender and higher foetal haemoglobin but not with alpha-thalassaemia or beta-globin haplotype. A tendency to familial clustering among elderly survivors did not reach statistical significance. Painful crises ameliorated with age and there was a benign course in pregnancy. Mean haemoglobin levels fell with age and were generally associated with rising creatinine levels indicating the importance of renal failure. Elderly survivors present some features of intrinsic mildness but also manifest age-related amelioration of painful crises and falling haemoglobin levels from progressive renal damage.

Garrick D, De Gobbi M, Gibbons R, Higgs DR. 2009. The regulatory interplay of CpG islands and nucleosome remodeling at mammalian primary response genes. Epigenomics, 1 (2), pp. 233.

Garrick D, De Gobbi M, Gibbons R, Higgs DR. 2009. XNP/ATRX at sites of nucleosome replacement. Epigenomics, 1 (2), pp. 233-234.

Vernimmen D, Marques-Kranc F, Sharpe JA, Sloane-Stanley JA, Wood WG, Wallace HA, Smith AJ, Higgs DR. 2009. Chromosome looping at the human alpha-globin locus is mediated via the major upstream regulatory element (HS -40). Blood, 114 (19), pp. 4253-4260. | Show Abstract | Read more

Previous studies in the mouse have shown that high levels of alpha-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human alpha cluster and their interactions with the alpha-globin promoter. All of these elements interact with the alpha-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human alpha globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS -40) results in loss of all the interactions and alpha-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human alpha-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.

Higgs DR, Weatherall DJ. 2009. The alpha thalassaemias. Cell Mol Life Sci, 66 (7), pp. 1154-1162. | Show Abstract | Read more

Recent work in the alpha thalassaemia field has started to provide some indication of the mechanisms involved in the very high frequency of the different forms of alpha thalassaemia among the populations of tropical countries, and, at the same time, is starting to define at least some of the mechanisms for its remarkable phenotypic heterogeneity. These diseases continue to provide extremely valuable models for the better understanding of the regulation of the alpha globin genes, and for human molecular pathology in general. The much less common disorders, ATR-16 and ATR-X are also providing valuable information about the spectrum of molecular lesions associated with different forms of mental retardation and about the molecular mechanisms involved in their varying phenotypes.

Haas PS, Roy NB, Gibbons RJ, Deville MA, Fisher C, Schwabe M, Bissé E, van Dorsselaer A, Higgs DR, Lübbert M. 2009. The role of X-inactivation in the gender bias of patients with acquired alpha-thalassaemia and myelodysplastic syndrome (ATMDS). Br J Haematol, 144 (4), pp. 538-545. | Show Abstract | Read more

Alpha thalassaemia myelodysplastic syndrome (ATMDS) is an unusual complication of chronic myeloid malignancy that is associated with a striking red cell phenotype. It represents an acquired form of alpha-thalassaemia that most commonly arises in the context of myelodysplasia. It has recently been shown that this condition occurs in association with somatic mutations of a known X-encoded trans-acting regulator of alpha globin gene (HBA) expression, ATRX. There is an unexplained, strong male preponderance of individuals with the ATMDS phenotype with a >5:1 male-female ratio and furthermore, all the somatic ATRX mutations described to date have been in males. Here we report the identification, in a single centre, of two females with ATMDS and mutations in the ATRX gene, proving that ATMDS associated with such mutations may occur, albeit rarely, in females. It seemed possible that females might be less likely to develop ATMDS if the inactivated copy of the ATRX gene (ATRX) became progressively re-activated throughout life. This study ruled out this hypothesis by investigating the pattern of ATRX inactivation in a cross-sectional analysis of normal females at ages ranging from newborn to 90 years.

Seah C, Levy MA, Jiang Y, Mokhtarzada S, Higgs DR, Gibbons RJ, Bérubé NG. 2008. Neuronal death resulting from targeted disruption of the Snf2 protein ATRX is mediated by p53. J Neurosci, 28 (47), pp. 12570-12580. | Show Abstract | Read more

ATRX, a chromatin remodeling protein of the Snf2 family, participates in diverse cellular functions including regulation of gene expression and chromosome alignment during mitosis and meiosis. Mutations in the human gene cause alpha thalassemia mental retardation, X-linked (ATR-X) syndrome, a rare disorder characterized by severe cognitive deficits, microcephaly and epileptic seizures. Conditional inactivation of the Atrx gene in the mouse forebrain leads to neonatal lethality and defective neurogenesis manifested by increased cell death and reduced cellularity in the developing neocortex and hippocampus. Here, we show that Atrx-null forebrains do not generate dentate granule cells due to a reduction in precursor cell number and abnormal migration of differentiating granule cells. In addition, fewer GABA-producing interneurons are generated that migrate from the ventral telencephalon to the cortex and hippocampus. Staining for cleaved caspase 3 demonstrated increased apoptosis in both the hippocampal hem and basal telencephalon concurrent with p53 pathway activation. Elimination of the tumor suppressor protein p53 in double knock-out mice rescued cell death in the embryonic telencephalon but only partially ameliorated the Atrx-null phenotypes at birth. Together, these findings show that ATRX deficiency leads to p53-dependent neuronal apoptosis which is responsible for some but not all of the phenotypic consequences of ATRX deficiency in the forebrain.

Garrick D, De Gobbi M, Samara V, Rugless M, Holland M, Ayyub H, Lower K, Sloane-Stanley J, Gray N, Koch C et al. 2008. The role of the polycomb complex in silencing alpha-globin gene expression in nonerythroid cells. Blood, 112 (9), pp. 3889-3899. | Show Abstract | Read more

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.

Rugless MJ, Fisher CA, Old JM, Sloane-Stanley J, Ayyub H, Higgs DR, Garrick D. 2008. A large deletion in the human alpha-globin cluster caused by a replication error is associated with an unexpectedly mild phenotype. Hum Mol Genet, 17 (19), pp. 3084-3093. | Show Abstract | Read more

We have characterized a newly identified 16.6 kb deletion which removes a significant proportion of the human alpha-globin cluster including the psizeta1, alpha(D), psialpha1 and alpha2-globin genes but leaves the duplicated alpha1 gene intact. This complicated rearrangement results from a combination of slippage and strand switching at sites of microhomology during replication. Functional analysis shows that expression of the remaining alpha1 gene is increased, rather than down-regulated by this deletion. This could be related to its proximity to the remote upstream alpha-globin regulatory elements or reduced competition for these elements in the absence of the dominant alpha2-globin gene. The finding of a very mild phenotype associated with such an extensive deletion in the alpha-globin cluster implies that much of the DNA removed by the deletion is likely to be functionally unimportant. These findings suggest that other than the upstream regulatory elements and promoter proximal elements there are unlikely to be additional positive cis-acting sequences in the alpha-globin cluster.

Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR et al. 2008. Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J Cell Biol, 182 (6), pp. 1083-1097. | Show Abstract | Read more

Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse alpha-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human alpha-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

Higgs DR, Wood WG. 2008. Genetic complexity in sickle cell disease. Proc Natl Acad Sci U S A, 105 (33), pp. 11595-11596. | Read more

Garrick D, De Gobbi M, Lynch M, Higgs DR. 2008. Switching genes on and off in haemopoiesis. Biochem Soc Trans, 36 (Pt 4), pp. 613-618. | Show Abstract | Read more

At present, the molecular mechanisms by which stem cells commit to and differentiate towards specific lineages are poorly characterized, and will need to be better understood before stem cells can be exploited fully in experimental and clinical settings. Transcriptional regulation, the ability to turn genes on and off, lies at the heart of these processes of lineage commitment and specification. We have focused on fully understanding how these decisions are made at a single mammalian gene locus, the alpha-globin genes, which become up-regulated in a tissue- and developmental-stage specific manner during haemopoiesis. The studies summarized in the present article have revealed that complete regulation of this gene cluster involves not only activating mechanisms in expressing erythroid cells, but also repressing mechanisms, involving the Polycomb complex and histone deacetylases which are present in non-erythroid tissues. Taken together, these observations provide a well-characterized model of how gene expression is fully regulated during the transition from stem cells through lineage commitment and terminal differentiation.

Higgs DR, Wood WG. 2008. Long-range regulation of alpha globin gene expression during erythropoiesis. Curr Opin Hematol, 15 (3), pp. 176-183. | Show Abstract | Read more

PURPOSE OF REVIEW: The analysis of globin gene expression during erythropoiesis has established many principles underlying normal mammalian gene expression. New aspects of gene regulation have been revealed by natural mutations that downregulate globin gene expression and cause thalassemia. Deletions involving sequences upstream of the alpha and beta clusters suggested that the globin genes might be controlled by remote regulatory elements. This was demonstrated experimentally and suggested that many mammalian genes may be controlled in a similar manner. RECENT FINDINGS: Completion of the Human Genome Project and the associated encyclopaedia of DNA elements (ENCODE) project confirmed that human gene expression is commonly controlled by long-range, cis-acting elements. The development of chromatin immunoprecipitation has allowed us to identify binding of transcription factors and chromatin modifications at the key cis-acting sequences in vivo. In addition, chromosome conformation capture has enabled us to address the topological models proposed to mediate long-range interactions. Together, these methods have given us some insight into how long-range elements may influence gene expression and how this process may be subverted in thalassemia. SUMMARY: The review asks how remote elements regulate alpha globin expression and how natural mutations interfere with this mechanism to cause alpha thalassemia. We also speculate as to why long-range control of gene expression may have evolved in higher organisms.

Higgs DR. 2008. A new dawn for stem-cell therapy. N Engl J Med, 358 (9), pp. 964-966. | Read more

dos Santos CO, Zhou S, Secolin R, Wang X, Cunha AF, Higgs DR, Kwiatkowski JL, Thein SL, Gallagher PG, Costa FF, Weiss MJ. 2008. Population analysis of the alpha hemoglobin stabilizing protein (AHSP) gene identifies sequence variants that alter expression and function. Am J Hematol, 83 (2), pp. 103-108. | Show Abstract | Read more

Alpha-hemoglobin stabilizing protein (AHSP) is a potential modifier of beta-thalassemia by virtue of its ability to detoxify excess free alpha-globin. However, examination of patients with beta-thalassemia from a few geographic regions failed to identify obvious AHSP mutations. We extended these studies by analyzing AHSP gene sequences in 366 anonymous individuals from five different areas of the world. We detected numerous polymorphisms comprising 18 different haplotypes and two rare missense mutations. Two sequence variations produce functional effects in laboratory assays. First, a rare missense mutation in a Brazilian/Mediterranean cohort converts asparagine to isoleucine at position 75 of AHSP protein and impairs its ability to inhibit reactive oxygen species production by alpha-hemoglobin. Second, a high-frequency polymorphism in intron 1 of the AHSP gene (12391 G>A) alters an Oct-1 transcription factor binding site previously shown to be important for optimal gene expression. The 12391A polymorphism impairs Oct-1 binding and inhibits the ability of AHSP regulatory sequences to activate expression of a linked luciferase reporter. Although structural mutations predicted to alter AHSP protein function or ablate its activity are rare, the 12391 G>A SNP is common and represents a potential mechanism through which genetically determined variations in AHSP expression could influence beta-thalassemia.

Vyas P, Higgs DR. 2008. Good news for the aging population? Blood, 112 (8), pp. 3001-3001. | Read more

Higgs DR, Vernimmen D, Wood B. 2008. Long-range regulation of alpha-globin gene expression. Adv Genet, 61 pp. 143-173. | Show Abstract | Read more

Over the past 20 years, there has been an increasing awareness that gene expression can be regulated by multiple cis-acting sequences located at considerable distances (10-1000 kb) from the genes they control. Detailed investigation of a few specialized mammalian genes, including the genes controlling the synthesis of hemoglobin, provide important models to understand how such long-range regulatory elements act. In general, these elements contain a high density of evolutionarily conserved, transcription factor-binding sites and in many ways resemble the upstream regulatory elements found adjacent to the promoters of genes in simpler organisms, differing only in the distance over which they act. We have investigated in detail how the remote regulatory elements of the alpha-globin cluster become activated as hematopoietic stem cells (HSCs) undergo commitment, lineage specification, and differentiation to form red blood cells. In turn, we have addressed how, during this process, the upstream elements control the correct spatial and temporal expression from the alpha-gene promoter which lies approximately 60 kb downstream of these elements. At present too few loci have been studied to determine whether there are general principles underlying long-range regulation but some common themes are emerging.

De Gobbi M, Anguita E, Hughes J, Sloane-Stanley JA, Sharpe JA, Koch CM, Dunham I, Gibbons RJ, Wood WG, Higgs DR. 2007. Tissue-specific histone modification and transcription factor binding in alpha globin gene expression. Blood, 110 (13), pp. 4503-4510. | Show Abstract | Read more

To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the alpha globin genes and 9 widely expressed genes flanking the alpha globin locus. This un-biased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human alpha globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo.

Steensma DP, Porcher JC, Hanson CA, Lathrop CL, Hoyer JD, Lasho TA, Tefferi A, Higgs DR. 2007. Prevalence of erythrocyte haemoglobin H inclusions in unselected patients with clonal myeloid disorders. Br J Haematol, 139 (3), pp. 439-442. | Show Abstract | Read more

Patients with clonal myeloid disorders, especially myelodysplastic syndromes (MDS), may acquire alpha-thalassaemia. To estimate the prevalence of this erythrocyte phenotype, we examined brilliant cresyl blue-stained blood smears from 201 patients with neoplastic myeloid disorders and 282 controls (195 non-clonal anaemia, 62 with medical illnesses without anaemia and 25 healthy persons). Haemoglobin H inclusions were detected in 8/100 patients with MDS (8%) and 2/81 (2.5%) patients with myeloproliferative disorders, but in none of the acute leukaemia patients or controls. We conclude that the emergence of thalassaemic clones may be relatively common in the disordered marrow milieu of MDS.

Argentaro A, Yang JC, Chapman L, Kowalczyk MS, Gibbons RJ, Higgs DR, Neuhaus D, Rhodes D. 2007. Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX. Proc Natl Acad Sci U S A, 104 (29), pp. 11939-11944. | Show Abstract | Read more

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

Giardine B, Riemer C, Hefferon T, Thomas D, Hsu F, Zielenski J, Sang Y, Elnitski L, Cutting G, Trumbower H et al. 2007. PhenCode: connecting ENCODE data with mutations and phenotype. Hum Mutat, 28 (6), pp. 554-562. | Show Abstract | Read more

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.

Muers MR, Sharpe JA, Garrick D, Sloane-Stanley J, Nolan PM, Hacker T, Wood WG, Higgs DR, Gibbons RJ. 2007. Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model. Am J Hum Genet, 80 (6), pp. 1138-1149. | Show Abstract | Read more

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.

Merryweather-Clarke AT, Atzberger A, Shah W, Woffendin HB, Soneji S, Waugh C, Clark K, Brown J, Sloane-Stanley J, Wood WG et al. 2007. Expression profile of healthy erythroid progenitors AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 571-571.

Vernimmen D, De Gobbi M, Sloane-Stanley JA, Wood WG, Higgs DR. 2007. Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression. EMBO J, 26 (8), pp. 2041-2051. | Show Abstract | Read more

To understand how mammalian genes are regulated from their natural chromosomal environment, we have analysed the molecular events occurring throughout a 150 kb chromatin segment containing the alpha globin gene locus as it changes from a poised, silent state in erythroid progenitors, to the fully activated state in late, erythroid cells. Active transcription requires the late recruitment of general transcription factors, mediator and Pol II not only to the promoter but also to its remote regulatory elements. Natural mutants of the alpha cluster show that whereas recruitment of the pre-initiation complex to the upstream elements occurs independently, recruitment to the promoter is largely dependent on the regulatory elements. An improved, quantitative chromosome conformation capture analysis demonstrates that this recruitment is associated with a conformational change, in vivo, apposing the promoter with its remote regulators, consistent with a chromosome looping mechanism. These findings point to a general mechanism by which a gene can be held in a poised state until the appropriate stage for expression, coordinating the level and timing of gene expression during terminal differentiation.

Serjeant GR, Higgs DR, Hambleton IR. 2007. Elderly survivors with homozygous sickle cell disease. N Engl J Med, 356 (6), pp. 642-643. | Read more

Wallace HA, Marques-Kranc F, Richardson M, Luna-Crespo F, Sharpe JA, Hughes J, Wood WG, Higgs DR, Smith AJ. 2007. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell, 128 (1), pp. 197-209. | Show Abstract | Read more

We have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse alpha globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human alpha globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human alpha thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases.

Higgs DR, Vernimmen D, Hughes J, Gibbons R. 2007. Using genomics to study how chromatin influences gene expression. Annu Rev Genomics Hum Genet, 8 (1), pp. 299-325. | Show Abstract | Read more

A postgenome challenge is to understand how the code in DNA is converted into the biological processes underlying various cell fates. By establishing the appropriate technical tools, we are moving from an era in which such questions have been asked by studying individual genes to one in which large domains, whole chromosomes, and the entire human genome can be investigated. These developments will allow us to study in parallel the transcriptional program and components of the epigenetic program (nuclear position, timing of replication, chromatin structure and modification, DNA methylation) to determine the hierarchy and order of events required to switch genes on and off during differentiation and development.

Ahmed MR, Chehal A, Zahed L, Taher A, Haidar J, Shamseddine A, O'Hea AM, Bienz N, Dgany O, Avidan N et al. 2006. Linkage and mutational analysis of the CDAN1 gene reveals genetic heterogeneity in congenital dyserythropoietic anemia type I. Blood, 107 (12), pp. 4968-4969. | Read more

De Gobbi M, Viprakasit V, Hughes JR, Fisher C, Buckle VJ, Ayyub H, Gibbons RJ, Vernimmen D, Yoshinaga Y, de Jong P et al. 2006. A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science, 312 (5777), pp. 1215-1217. | Show Abstract | Read more

We describe a pathogenetic mechanism underlying a variant form of the inherited blood disorder alpha thalassemia. Association studies of affected individuals from Melanesia localized the disease trait to the telomeric region of human chromosome 16, which includes the alpha-globin gene cluster, but no molecular defects were detected by conventional approaches. After resequencing and using a combination of chromatin immunoprecipitation and expression analysis on a tiled oligonucleotide array, we identified a gain-of-function regulatory single-nucleotide polymorphism (rSNP) in a nongenic region between the alpha-globin genes and their upstream regulatory elements. The rSNP creates a new promoterlike element that interferes with normal activation of all downstream alpha-like globin genes. Thus, our work illustrates a strategy for distinguishing between neutral and functionally important rSNPs, and it also identifies a pathogenetic mechanism that could potentially underlie other genetic diseases.

Ahmed MR, Zaki M, Sabry MA, Higgs D, Vyas P, Wood W, Wickramasinghe SN. 2006. Evidence of genetic heterogeneity in congenital dyserythropoietic anaemia type I. Br J Haematol, 133 (4), pp. 444-445. | Read more

Costa DB, Fisher CA, Miller KB, Pihan GA, Steensma DP, Gibbons RJ, Higgs DR. 2006. 5-Azacytidine treatment of the patient with ATMDS (vol 76, pg 432, 2006) EUROPEAN JOURNAL OF HAEMATOLOGY, 76 (5), pp. 453-453. | Read more

Viprakasit V, Harteveld CL, Ayyub H, Stanley JS, Giordano PC, Wood WG, Higgs DR. 2006. A novel deletion causing alpha thalassemia clarifies the importance of the major human alpha globin regulatory element. Blood, 107 (9), pp. 3811-3812. | Read more

Costa DB, Fisher CA, Miller KB, Pihan GA, Steensma DP, Gibbons RJ, Higgs DR. 2006. A novel mutation in the last exon of ATRX in a patient with alpha-thalassemia myelodysplastic syndrome. Eur J Haematol, 76 (5), pp. 432-453. | Show Abstract | Read more

We describe a patient with acquired alpha-thalassemia myelodysplastic syndrome (ATMDS). A previously healthy 66-year-old man presented with hemoglobin of 9.3 g/dL, mean corpuscular volume 59 fL, and a bone marrow aspirate with increased erythroid precursors and hypolobulated megakaryocytes. Hemoglobin H inclusions were seen in most red cells after 1% brilliant cresyl blue supravital stain of the peripheral blood. At the molecular level, we identified of a novel mutation in the most 3' exon of the ATRX gene (CGA-->TGA substitution in codon 2407) resulting in a premature termination codon (p.R2407X). This case provides further evidence for a link between ATRX mutations and ATMDS, and suggests a possible role for the conserved Q-box element in ATRX function.

Garrick D, Sharpe JA, Arkell R, Dobbie L, Smith AJ, Wood WG, Higgs DR, Gibbons RJ. 2006. Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues. PLoS Genet, 2 (4), pp. e58. | Show Abstract | Read more

ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.

Solberg LC, Valdar W, Gauguier D, Nunez G, Taylor A, Burnett S, Arboledas-Hita C, Hernandez-Pliego P, Davidson S, Burns P et al. 2006. A protocol for high-throughput phenotyping, suitable for quantitative trait analysis in mice. Mamm Genome, 17 (2), pp. 129-146. | Show Abstract | Read more

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.

Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J, Wood WG, Higgs DR, Iborra FJ, Buckle VJ. 2006. Coregulated human globin genes are frequently in spatial proximity when active. J Cell Biol, 172 (2), pp. 177-187. | Show Abstract | Read more

The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated alpha- and beta-globin genes and show that the gene-dense chromatin surrounding the human alpha-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active alpha- and beta-globin genes and of homologous alpha-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human beta-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.

Higgs DR, Vernimmen D, De Gobbi M, Anguita E, Hughes J, Buckle V, Iborra F, Garrick D, Wood WG. 2006. How transcriptional and epigenetic programmes are played out on an individual mammalian gene cluster during lineage commitment and differentiation. Biochem Soc Symp, 73 (73), pp. 11-22. | Show Abstract | Read more

In the post-genomic era, a great deal of work has focused on understanding how DNA sequence is used to programme complex nuclear, cellular and tissue functions throughout differentiation and development. There are many approaches to these issues, but we have concentrated on understanding how a single mammalian gene cluster is activated or silenced as stem cells undergo lineage commitment, differentiation and maturation. In particular we have analysed the alpha globin cluster, which is expressed in a cell-type- and developmental stage-specific manner in the haemopoietic system. Our studies include analysis of the transcriptional programme that accompanies globin gene activation, focusing on the expression of relevant transcription factors and cofactors. Binding of these factors to the chromosomal domain containing the alpha globin cluster has been characterized by ChIP (chromatin immunoprecipitation). In addition, we have monitored the epigenetic modifications (e.g. nuclear position, timing of replication, chromatin modification, DNA methylation) that occur as the genes are activated (in erythroid cells) or silenced (e.g. in granulocytes) as haemopoiesis proceeds. Together, these observations provide a uniquely well-characterized model illustrating the mechanisms that regulate and memorize patterns of mammalian gene expression as stem cells undergo lineage specification, differentiation and terminal maturation.

Garrick D, Sharpe JA, Arkell R, Dobbie L, Smith AJ, Wood WG, Higgs DR, Gibbons RJ. 2006. Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues. PLoS genetics, 2 (4), | Show Abstract | Read more

ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.

Weatherall MW, Higgs DR, Weiss H, Weatherall DJ, Serjeant GR. 2005. Phenotype/genotype relationships in sickle cell disease: a pilot twin study. Clin Lab Haematol, 27 (6), pp. 384-390. | Show Abstract | Read more

The roles of genetic and non-genetic factors in the haematology, growth and clinical features of sickle cell disease have been studied in nine identical twin pairs (six homozygous sickle cell disease, three sickle cell-haemoglobin C disease). A comparison group of 350 age-gender matched sibling pairs, selected to have an age difference of <5 years, was used for assessing the concordance of numerical data. Attained height, weight at attained height, fetal haemoglobin, total haemoglobin, mean cell volume, mean cell haemoglobin and total bilirubin levels showed significantly greater correlation in identical twins than in siblings. Twins showed similarities in the prevalence and degree of splenomegaly, susceptibility to priapism, and in onset of menarche, but other clinical complications were discordant in prevalence and severity. These findings suggest that physical growth and many haematological characteristics are subject to genetic influences, but that non-genetic factors contribute to the variance in disease manifestations.

Hughes JR, Cheng JF, Ventress N, Prabhakar S, Clark K, Anguita E, De Gobbi M, de Jong P, Rubin E, Higgs DR. 2005. Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences. Proc Natl Acad Sci U S A, 102 (28), pp. 9830-9835. | Show Abstract | Read more

An important step toward improving the annotation of the human genome is to identify cis-acting regulatory elements from primary DNA sequence. One approach is to compare sequences from multiple, divergent species. This approach distinguishes multispecies conserved sequences (MCS) in noncoding regions from more rapidly evolving neutral DNA. Here, we have analyzed a region of approximately 238kb containing the human alpha globin cluster that was sequenced and/or annotated across the syntenic region in 22 species spanning 500 million years of evolution. Using a variety of bioinformatic approaches and correlating the results with many aspects of chromosome structure and function in this region, we were able to identify and evaluate the importance of 24 individual MCSs. This approach sensitively and accurately identified previously characterized regulatory elements but also discovered unidentified promoters, exons, splicing, and transcriptional regulatory elements. Together, these studies demonstrate an integrated approach by which to identify, subclassify, and predict the potential importance of MCSs.

Bérubé NG, Mangelsdorf M, Jagla M, Vanderluit J, Garrick D, Gibbons RJ, Higgs DR, Slack RS, Picketts DJ. 2005. The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis. J Clin Invest, 115 (2), pp. 258-267. | Show Abstract | Read more

Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however, the role of these proteins in normal CNS development is unknown. Here, we used a conditional gene-targeting approach to inactivate Atrx, specifically in the forebrain of mice. Loss of ATRX protein caused widespread hypocellularity in the neocortex and hippocampus and a pronounced reduction in forebrain size. Neuronal "birthdating" confirmed that fewer neurons reached the superficial cortical layers, despite normal progenitor cell proliferation. The loss of cortical mass resulted from a 12-fold increase in neuronal apoptosis during early stages of corticogenesis in the mutant animals. Moreover, cortical progenitors isolated from Atrx-null mice undergo enhanced apoptosis upon differentiation. Taken together, our results indicate that ATRX is a critical mediator of cell survival during early neuronal differentiation. Thus, increased neuronal loss may contribute to the severe mental retardation observed in human patients.

Steensma DP, Gibbons RJ, Higgs DR. 2005. Acquired alpha-thalassemia in association with myelodysplastic syndrome and other hematologic malignancies. Blood, 105 (2), pp. 443-452. | Show Abstract | Read more

Abnormalities of hemoglobin synthesis are usually inherited but may also arise as a secondary manifestation of another disease, most commonly hematologic neoplasia. Acquired hemoglobin disorders can be seen in any population and are not restricted to areas of the world with high incidences of inherited hemoglobinopathies. In fact, the acquired hemoglobinopathies may be more readily recognized where inherited hemoglobin abnormalities are rare and less likely to cause diagnostic confusion. Acquired alpha-thalassemia is the best characterized of the acquired red blood cell disorders in patients with hematologic malignancy, and it is almost always associated with a myelodysplastic syndrome (MDS). At least 2 molecular mechanisms for acquired alpha-thalassemia are now recognized: acquired deletion of the alpha-globin gene cluster limited to the neoplastic clone and, more commonly, inactivating somatic mutations of the trans-acting chromatin-associated factor ATRX, which cause dramatic down-regulation of alpha-globin gene expression. Here we review the clinical, hematologic, and molecular genetic features of alpha-thalassemia arising in a clonal myeloid disorder, and we discuss howATRX might affect gene expression in normal and abnormal hematopoiesis through epigenetic mechanisms.

Steensma DP, Gibbons RJ, Mesa RA, Tefferi A, Higgs DR. 2005. Somatic point mutations in RUNX1/CBFA2/AML1 are common in high-risk myelodysplastic syndrome, but not in myelofibrosis with myeloid metaplasia. Eur J Haematol, 74 (1), pp. 47-53. | Show Abstract | Read more

OBJECTIVE: Acquired somatic point mutations in RUNX1/CBFA2/AML1 have recently been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Given the importance of core-binding factor in megakaryocytic differentiation and platelet production, as well as the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), we hypothesised that RUNX1 gene mutations might be common in MMM. In addition, it is unclear whether patients with MDS-associated acquired alpha thalassaemia (ATMDS), a special subgroup with a very high incidence of point mutations in the ATRX gene, have an especially high incidence of RUNX1 mutations. METHODS: We analysed samples from 78 patients for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC): 26 with MMM and 52 with MDS, including 18 with ATMDS. RESULTS: We found five RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 or a history of treated AML, but none in MMM patients. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18, 11.1%) when compared with MDS without thalassaemia (3/34, 8.8%; P = 0.58). CONCLUSION: RUNX1 point mutations are common in high-risk MDS, but not in MMM. DHPLC is a useful technique for high-throughput analysis of RUNX1 mutation status in myeloid disorders, and may be complementary to screening via other methods.

Price TS, Regan R, Mott R, Hedman A, Honey B, Daniels RJ, Smith L, Greenfield A, Tiganescu A, Buckle V et al. 2005. SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data. Nucleic Acids Res, 33 (11), pp. 3455-3464. | Show Abstract | Read more

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.

Higgs DR, Garrick D, Anguita E, De Gobbi M, Hughes J, Muers M, Vernimmen D, Lower K, Law M, Argentaro A et al. 2005. Understanding alpha-globin gene regulation: Aiming to improve the management of thalassemia. Ann N Y Acad Sci, 1054 (1), pp. 92-102. | Show Abstract | Read more

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.

Anguita E, Hughes J, Heyworth C, Blobel GA, Wood WG, Higgs DR. 2004. Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2. EMBO J, 23 (14), pp. 2841-2852. | Show Abstract | Read more

How does an emerging transcriptional programme regulate individual genes as stem cells undergo lineage commitment, differentiation and maturation? To answer this, we have analysed the dynamic protein/DNA interactions across 130 kb of chromatin containing the mouse alpha-globin cluster in cells representing all stages of differentiation from stem cells to mature erythroblasts. The alpha-gene cluster appears to be inert in pluripotent cells, but priming of expression begins in multipotent haemopoietic progenitors via GATA-2. In committed erythroid progenitors, GATA-2 is replaced by GATA-1 and binding is extended to additional sites including the alpha-globin promoters. Both GATA-1 and GATA-2 nucleate the binding of various protein complexes including SCL/LMO2/E2A/Ldb-1 and NF-E2. Changes in protein/DNA binding are accompanied by sequential alterations in long-range histone acetylation and methylation. The recruitment of polymerase II, which ultimately leads to a rapid increase in alpha-globin transcription, occurs late in maturation. These studies provide detailed evidence for the more general hypothesis that commitment and differentiation are primarily driven by the sequential appearance of key transcriptional factors, which bind chromatin at specific, high-affinity sites.

Viprakasit V, Tanphaichitr VS, Chinchang W, Sangkla P, Weiss MJ, Higgs DR. 2004. Evaluation of alpha hemoglobin stabilizing protein (AHSP) as a genetic modifier in patients with beta thalassemia. Blood, 103 (9), pp. 3296-3299. | Show Abstract | Read more

Although beta thalassemia is considered to be a classic monogenic disease, it is clear that there is considerable clinical variability between patients who inherit identical beta globin gene mutations, suggesting that there may be a variety of genetic determinants influencing different clinical phenotypes. It has been suggested that variations in the structure or amounts of a highly expressed red cell protein (alpha hemoglobin stabilizing protein [AHSP]), which can stabilize free alpha globin chains in vitro, could influence disease severity in patients with beta thalassemia. To address this hypothesis, we studied 120 patients with Hb E-beta thalassemia with mild, moderate, or severe clinical phenotypes. Using gene mapping, direct genomic sequencing, and extended haplotype analysis, we found no mutation or specific association between haplotypes of AHSP and disease severity in these patients, suggesting that AHSP is not a disease modifier in Hb E-beta thalassemia. It remains to be seen if any association between AHSP and clinical severity is present in other population groups with a high frequency of beta thalassemia.

Tufarelli C, Hardison R, Miller W, Hughes J, Clark K, Ventress N, Frischauf AM, Higgs DR. 2004. Comparative analysis of the alpha-like globin clusters in mouse, rat, and human chromosomes indicates a mechanism underlying breaks in conserved synteny. Genome Res, 14 (4), pp. 623-630. | Show Abstract | Read more

We have sequenced and fully annotated a 65,871-bp region of mouse Chromosome 17 including the Hba-ps4 alpha-globin pseudogene. Comparative sequence analysis with the functional alpha-globin loci at human Chromosome 16p13.3 and mouse Chromosome 11 shows that this segment of mouse Chromosome 17 contains a group of three alpha-like pseudogenes (Hba-psm-Hba-ps4-Hba-q3), similar to the duplicated sets found at the functional mouse cluster on Chromosome 11. In addition, exons 7 to 12 of the mLuc7L gene are present just downstream from the pseudogene cluster, indicating that this clone contains the region in which human 16p13.3 switches in synteny between mouse Chromosomes 11 and 17. Comparison of the sequences around the alpha-like clusters on the two mouse chromosomes reveals the presence of conserved tandem repeats. We propose that these repetitive elements have played a role in the fragmentation of the mouse alpha cluster during evolution.

Steensma DP, Higgs DR, Fisher CA, Gibbons RJ. 2004. Acquired somatic ATRX mutations in myelodysplastic syndrome associated with alpha thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations. Blood, 103 (6), pp. 2019-2026. | Show Abstract | Read more

Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.

Steensma DP, Viprakasit V, Hendrick A, Goff DK, Leach J, Gibbons RJ, Higgs DR. 2004. Deletion of the alpha-globin gene cluster as a cause of acquired alpha-thalassemia in myelodysplastic syndrome. Blood, 103 (4), pp. 1518-1520. | Show Abstract | Read more

Rarely, myelodysplastic syndrome (MDS) is complicated by an acquired form of alpha-thalassemia (alpha-thalassemia in myelodysplastic syndrome [ATMDS]) characterized by hypochromic, microcytic, anisopoikilocytic red blood cells with hemoglobin H (HbH) inclusions. Acquired mutations in ATRX, a chromatin remodeling gene, have recently been found in 12 patients with typical features of ATMDS, though they have not been detected in MDS patients with similar red blood cell findings but little HbH. The alpha-globin genes themselves have appeared normal in all ATMDS patients studied to date. Here we characterize the molecular defect in a unique MDS patient with rare HbH inclusions in which an abnormal clone lost a greater than 1.9-Mb segment of the telomeric region of the short arm of one allele of chromosome 16, including both alpha-globin genes. Red blood cell changes associated with this acquired somatic genotype (--/alpha alpha) are surprisingly severe, demonstrating that a minor globin chain imbalance may be unexpectedly deleterious during the abnormal erythropoiesis that occurs in the context of MDS.

Garrick D, Samara V, McDowell TL, Smith AJ, Dobbie L, Higgs DR, Gibbons RJ. 2004. A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain. Gene, 326 (1-2), pp. 23-34. | Show Abstract | Read more

Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.

Alexander N, Higgs D, Dover G, Serjeant GR. 2004. Are there clinical phenotypes of homozygous sickle cell disease? Br J Haematol, 126 (4), pp. 606-611. | Show Abstract | Read more

The distribution of clinical features was examined in subjects with homozygous sickle cell (SS) disease in the Jamaican Cohort Study to determine whether there is evidence of distinct clustering of symptoms or clinical phenotypes. A twofold model yielded groups that could be interpreted as painful crisis or leg ulcer phenotypes and 78% of patients were classified with 95% confidence into one of these. The painful crisis phenotype also manifested higher frequencies of dactylitis, meningitis/septicaemia, acute chest syndrome and stroke. Attempts to define a three-group model were less convincing although 43% of patients could be allocated with 95% confidence. The three-group model essentially divided subjects with the leg ulcer phenotype into subgroups with higher and lower frequencies of painful crisis, dactylitis, meningitis/septicaemia and acute chest syndrome. In the three-group model, the painful crisis phenotype had lower total haemoglobin, fetal haemoglobin, mean cell volume and higher reticulocytes but there was no apparent influence of alpha thalassaemia or beta globin haplotype. Both environmental and genetic factors are likely to contribute to most manifestations of SS disease and the evidence for different clinical phenotypes suggests that a search for associated genetic polymorphisms may provide insights into the mechanisms of clinical variability in SS disease.

Viprakasit V, Tanphaichitr VS, Veerakul G, Chinchang W, Petrarat S, Pung-Amritt P, Higgs DR. 2004. Co-inheritance of Hb Pak Num Po, a novel alpha1 gene mutation, and alpha0 thalassemia associated with transfusion-dependent Hb H disease. Am J Hematol, 75 (3), pp. 157-163. | Show Abstract | Read more

Hb H disease is generally associated with moderate to severe anemia but rarely requires regular blood transfusion. We recently studied two apparently unrelated patients with transfusion-dependent Hb H disease. Hemoglobin studies demonstrated Hb H and Hb Bart's without other detectable abnormal globin species. Extensive molecular analyses of the alpha globin genes and their regulatory sequence (HS-40) revealed that both patients are compound heterozygotes for alpha0 thalassemia (--(SEA)) and a novel point mutation, a thymidine insertion after codon 131 of the alpha1 gene. The resulting frameshift gives rise to a highly unstable alpha globin chain, which we refer to as "Hb Pak Num Po," containing an additional 34 amino acids. This unusual alpha1 globin variant clearly causes alpha thalassemia, but the unexpectedly severe phenotype suggests that this mutation may have additional effects on red cell physiology. A PCR-based (ARMS) assay was developed for rapid detection of this novel mutation, and this might be useful to study the prevalence of this novel mutation which poses potentially significant clinical consequences in populations of Southeast Asia. Detecting carriers of this mutation using the molecular diagnostic procedures described will provide the means to screen and prevent a potentially severe form of alpha thalassemia in Thailand.

Higgs DR. 2004. Gene regulation in hematopoiesis: new lessons from thalassemia. Hematology Am Soc Hematol Educ Program, pp. 1-13. | Show Abstract | Read more

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 alpha and beta 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.

Gibbons RJ, Pellagatti A, Garrick D, Wood WG, Malik N, Ayyub H, Langford C, Boultwood J, Wainscoat JS, Higgs DR. 2003. Identification of acquired somatic mutations in the gene encoding chromatin-remodeling factor ATRX in the alpha-thalassemia myelodysplasia syndrome (ATMDS). Nat Genet, 34 (4), pp. 446-449. | Show Abstract | Read more

Inherited mutations of specific genes have elucidated the normal roles of the proteins they encode by relating specific mutations to particular phenotypes. But many potentially informative mutations in such genes are lethal early in development. Consequently, inherited mutations may not reflect all the functional roles of such proteins. Acquired, somatic defects should reflect a wider spectrum of mutations because they are not prone to negative selection in development. It has been difficult to identify such mutations so far, but microarray analysis provides a new opportunity to do so. Using this approach, we have shown that in individuals with myelodysplasia associated with alpha-thalassemia (ATMDS), somatic mutations of the gene encoding the chromatin remodeling factor ATRX cause an unexpectedly severe hematological phenotype compared with the wide spectrum of inherited mutations affecting this gene. These findings cast new light on this pleiotropic cofactor, which appears to be an essential component rather than a mere facilitator of globin gene expression.

Tufarelli C, Stanley JA, Garrick D, Sharpe JA, Ayyub H, Wood WG, Higgs DR. 2003. Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat Genet, 34 (2), pp. 157-165. | Show Abstract | Read more

Nearly all human genetic disorders result from a limited repertoire of mutations in an associated gene or its regulatory elements. We recently described an individual with an inherited form of anemia (alpha-thalassemia) who has a deletion that results in a truncated, widely expressed gene (LUC7L) becoming juxtaposed to a structurally normal alpha-globin gene (HBA2). Although it retains all of its local and remote cis-regulatory elements, expression of HBA2 is silenced and its CpG island becomes completely methylated early during development. Here we show that in the affected individual, in a transgenic model and in differentiating embryonic stem cells, transcription of antisense RNA mediates silencing and methylation of the associated CpG island. These findings identify a new mechanism underlying human genetic disease.

Brownlie A, Hersey C, Oates AC, Paw BH, Falick AM, Witkowska HE, Flint J, Higgs D, Jessen J, Bahary N et al. 2003. Characterization of embryonic globin genes of the zebrafish. Dev Biol, 255 (1), pp. 48-61. | Show Abstract | Read more

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.

Viprakasit V, Kidd AM, Ayyub H, Horsley S, Hughes J, Higgs DR. 2003. De novo deletion within the telomeric region flanking the human alpha globin locus as a cause of alpha thalassaemia. Br J Haematol, 120 (5), pp. 867-875. | Show Abstract | Read more

We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.

Xue Y, Gibbons R, Yan Z, Yang D, McDowell TL, Sechi S, Qin J, Zhou S, Higgs D, Wang W. 2003. The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies. Proc Natl Acad Sci U S A, 100 (19), pp. 10635-10640. | Show Abstract | Read more

ATRX syndrome is characterized by X-linked mental retardation associated with alpha-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.

Anguita E, Sharpe JA, Sloane-Stanley JA, Tufarelli C, Higgs DR, Wood WG. 2002. Deletion of the mouse alpha-globin regulatory element (HS -26) has an unexpectedly mild phenotype. Blood, 100 (10), pp. 3450-3456. | Show Abstract | Read more

Natural deletions of the region upstream of the human alpha-globin gene cluster, together with expression studies in cell lines and transgenic mice, identified a single element (HS -40) as necessary and perhaps sufficient for high-level expression of the alpha-globin genes. A similar element occupies the corresponding position upstream of the mouse (m) alpha-globin genes (mHS -26) and was thought to have similar functional properties. We knocked out mHS -26 by homologous recombination and observed the surprising result that instead of the expected severe alpha-thalassemia phenotype, the mice had a mild disease. Transcription levels of the mouse genes were reduced by about 50%, but homozygotes were healthy, with normal hemoglobin levels and only mild decreases in mean corpuscular volume and mean corpuscular hemoglobin. These results may indicate differences in the regulation of the alpha-globin clusters in mice and humans or that additional cis-acting elements remain to be characterized in one or both clusters.

Viprakasit V, Green S, Height S, Ayyub H, Higgs DR. 2002. Hb H hydrops fetalis syndrome associated with the interaction of two common determinants of alpha thalassaemia (--MED/(alpha)TSaudi(alpha)). Br J Haematol, 117 (3), pp. 759-762. | Show Abstract | Read more

To date, more than 35 single or oligonucleotide mutations of the alpha genes that cause alpha thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of alpha thalassaemia. Here we show for the first time that the interaction between two relatively common forms of alpha thalassaemia (--MED/(alpha)TSaudi(alpha)) may be associated with a clinically severe form of alpha thalassaemia, Hb H hydrops fetalis.

Viprakasit V, Tanphaichitr VS, Pung-Amritt P, Petrarat S, Suwantol L, Fisher C, Higgs DR. 2002. Clinical phenotypes and molecular characterization of Hb H-Paksé disease. Haematologica, 87 (2), pp. 117-125. | Show Abstract

BACKGROUND AND OBJECTIVES: Hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (TAA-->CAA) in the a2 gene, is the most common non-deletional type of a thalassemia in Southeast Asia. This mutation can most easily be detected by loss of an MseI-restriction site (T/TAA) spanning the termination codon. Recently, we sequenced the a globin genes from patients with a thalassemia in whom this MseI site was absent. This revealed, a previously described termination codon mutation (TAA-->TAT) associated with Hb Paksé. This prompted us to re-evaluate the molecular basis of a thalassaemia in other Thai patients with non-deletional types of Hb H disease. DESIGN AND METHODS: DNA samples from 30 patients, previously diagnosed as having Hb H-CS disease, were characterized by direct genomic sequencing and by using a mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical and hematologic data were assessed. RESULTS: Hemoglobin electrophoresis in almost all 30 unrelated patients with non-deletional a thalassemia revealed a slow migrating band resembling Hb CS. Five of these patients were found to have Hb H-Paksé disease and the remainder had Hb H-CS disease. Comparing the hematology in patients with these two genotypes, no significant differences were found except that the proportion of Hb H was higher in patients with Hb H-Paksé disease. INTERPRETATION AND CONCLUSIONS: These results suggest that termination codon mutations may have been previously misidentified in many cases of non-deletional Hb H disease. Findings from six unrelated families described in this study suggest that the proportion of patients with the Hb Paksé mutation might be underestimated and that this mutation could be prevalent in Southeast Asia. Analysis of mismatched-PCR-RFLP, described here, was shown to provide an unequivocal diagnosis and will be applicable in population screening programs.

Anguita E, Jonhson CA, Wood WG, Turner BM, Higgs DR. 2001. Identification of a conserved erythroid specific domain of histone acetylation across the a globin gene cluster. BLOOD, 98 (11), pp. 437A-437A.

Viprakasit V, Gibbons RJ, Broughton BC, Lehmann AR, Higgs DR. 2001. Mutations in a general transcription factor, TFIIH result in a novel unlinked form of beta thalassaemia in individuals with trichothiodystrophy. BLOOD, 98 (11), pp. 438A-438A.

Viprakasit V, Gibbons RJ, Broughton BC, Tolmie JL, Brown D, Lunt P, Winter RM, Marinoni S, Stefanini M, Brueton L et al. 2001. Mutations in the general transcription factor TFIIH result in beta-thalassaemia in individuals with trichothiodystrophy. Hum Mol Genet, 10 (24), pp. 2797-2802. | Show Abstract | Read more

The transcription factor TFIIH is involved in both basal transcription and DNA repair. Mutations in the XPD helicase component of TFIIH can result in the diverse clinical features associated with xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). It is generally believed that the multi-system abnormalities associated with TTD are the result of a subtle deficiency in basal transcription. However, to date, there has been no clear demonstration of a defect in expression of any specific gene in individuals with these syndromes. Here we show that the specific mutations in XPD that cause TTD result in reduced expression of the beta-globin genes in these individuals. Eleven TTD patients with characterized mutations in the XPD gene have the haematological features of beta-thalassaemia trait, and reduced levels of beta-globin synthesis and beta-globin mRNA. All these parameters were normal in three patients with XP. These findings provide the first evidence for reduced expression of a specific gene in TTD. They support the hypothesis that many of the clinical features of TTD result from inadequate expression of a diverse set of highly expressed genes.

Anguita E, Johnson CA, Wood WG, Turner BM, Higgs DR. 2001. Identification of a conserved erythroid specific domain of histone acetylation across the alpha-globin gene cluster. Proc Natl Acad Sci U S A, 98 (21), pp. 12114-12119. | Show Abstract | Read more

We have analyzed the pattern of core histone acetylation across 250 kb of the telomeric region of the short arm of human chromosome 16. This gene-dense region, which includes the alpha-globin genes and their regulatory elements embedded within widely expressed genes, shows marked differences in histone acetylation between erythroid and non-erythroid cells. In non-erythroid cells, there was a uniform 2- to 3-fold enrichment of acetylated histones, compared with heterochromatin, across the entire region. In erythroid cells, an approximately 100-kb segment of chromatin encompassing the alpha genes and their remote major regulatory element was highly enriched in histone H4 acetylated at Lys-5. Other lysines in the N-terminal tail of histone H4 showed intermediate and variable levels of enrichment. Similar broad segments of erythroid-specific histone acetylation were found in the corresponding syntenic regions containing the mouse and chicken alpha-globin gene clusters. The borders of these regions of acetylation are located in similar positions in all three species, and a sharply defined 3' boundary coincides with the previously identified breakpoint in conserved synteny between these species. We have therefore demonstrated that an erythroid-specific domain of acetylation has been conserved across several species, encompassing not only the alpha-globin genes but also a neighboring widely expressed gene. These results contrast with those at other clusters and demonstrate that not all genes are organized into discrete regulatory domains.

Flint J, Tufarelli C, Peden J, Clark K, Daniels RJ, Hardison R, Miller W, Philipsen S, Tan-Un KC, McMorrow T et al. 2001. Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster. Hum Mol Genet, 10 (4), pp. 371-382. | Show Abstract | Read more

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Daniels RJ, Peden JF, Lloyd C, Horsley SW, Clark K, Tufarelli C, Kearney L, Buckle VJ, Doggett NA, Flint J, Higgs DR. 2001. Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16. Hum Mol Genet, 10 (4), pp. 339-352. | Show Abstract | Read more

We have sequenced 1949 kb from the terminal Giemsa light band of human chromosome 16p, enabling us to fully annotate the region extending from the telomeric repeats to the previously published tuberous sclerosis disease 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes. This region can be subdivided into two GC-rich, Alu-rich domains and one GC-rich, Alu-poor domain. The entire region is extremely gene rich, containing 100 confirmed genes and 20 predicted genes. Many of the genes encode widely expressed proteins orchestrating basic cellular processes (e.g. DNA recombination, repair, transcription, RNA processing, signal transduction, intracellular signalling and mRNA translation). Others, such as the alpha globin genes (HBA1 and HBA2), PDIP and BAIAP3, are specialized tissue-restricted genes. Some of the genes have been previously implicated in the pathophysiology of important human genetic diseases (e.g. asthma, cataracts and the ATR-16 syndrome). Others are known disease genes for alpha thalassaemia, adult polycystic kidney disease and tuberous sclerosis. There is also linkage evidence for bipolar affective disorder, epilepsy and autism in this region. Sixty-three chromosomal deletions reported here and elsewhere allow us to interpret the results of removing progressively larger numbers of genes from this well defined human telomeric region.

Tufarelli C, Frischauf AM, Hardison R, Flint J, Higgs DR. 2001. Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain. Genomics, 71 (3), pp. 307-314. | Show Abstract | Read more

We have identified the first gene lying on the centromeric side of the alpha-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the alpha-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5' splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the alpha-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse alpha-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human alpha-globin cluster to within an 8-kb segment of chromosome 16.

Horsley SW, Daniels RJ, Anguita E, Raynham HA, Peden JF, Villegas A, Vickers MA, Green S, Waye JS, Chui DH et al. 2001. Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects. Eur J Hum Genet, 9 (3), pp. 217-225. | Show Abstract | Read more

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

Villegas A, Anguita E, Noguera N, González FA, Ropero P, Sánchez J, Milani AC, Espinos D, Monash DB, Higgs D. 2000. A new polymorphim (G->A) in the psizeta1 globin gene. Haematologica, 85 (9), pp. 899-901. | Show Abstract

BACKGROUND AND OBJECTIVES: a-globin cluster polymorphisms are obtained with specific restriction enzymes (Xba I, Eco RI, Sac I, Apa I, Bgl II, etc) that can also have implications for genetic analysis. DESIGN AND AND METHODS: We studied three unrelated patients; one from Argentina, one from Spain and one from Australia but of Polish origin. Genomic DNA was digested with several different restriction enzymes and probes, amplified and sequenced with an ABI Prism 310 sequencer. RESULTS: In the three patients an abnormal 26 kb band appeared when they were studied with restriction enzyme Bgl II and z probe. A fragment of 944 bp was amplified with primers that cover from -280 to +714 bp of the recognition sequence of Bgl II enzyme (AGATCT) localized 5' from pseudogene z1. After digestion of this PCR product with Bgl II, two fragments of 714 and 280 bp were produced in normal controls, whereas in patient #1 the PCR fragment was undigested and in patients 2 and 3 both undigested and digested fragments were observed. Sequencing of the PCR fragment showed that in all three patients it was the same polymorphism (G->A) at nucleotide 153171 of the 16 p sequence found in the Bgl II recognition site that changed to AAATCT. INTERPRETATION AND CONCLUSIONS: We describe a new polymorphism in the yz1 first exon Bgl II restriction site (G->A). The polymorphism is associated in cis with haplotype -a3.7. The fragment obtained by PCR enabled us to corroborate the presence of the polymorphism quickly without having to use complicated sequencing techniques.

Barbour VM, Tufarelli C, Sharpe JA, Smith ZE, Ayyub H, Heinlein CA, Sloane-Stanley J, Indrak K, Wood WG, Higgs DR. 2000. alpha-thalassemia resulting from a negative chromosomal position effect. Blood, 96 (3), pp. 800-807. | Show Abstract

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

Guerrini R, Shanahan JL, Carrozzo R, Bonanni P, Higgs DR, Gibbons RJ. 2000. A nonsense mutation of the ATRX gene causing mild mental retardation and epilepsy. Ann Neurol, 47 (1), pp. 117-121. | Show Abstract | Read more

Mutations in the X-encoded gene ATRX are known to give rise to profound syndromal mental retardation (MR). Here, we describe a pedigree, including 4 affected family members with a 324C-->T nonsense mutation in the ATRX gene. Although 2 patients have moderate to profound MR and the typical facial features of ATR-X syndrome, the other 2 patients presented with mild MR and epilepsy but without the characteristic facial dysmorphism. Mutations in the ATRX gene should be considered as a cause of mild MR in male patients lacking specific diagnostic features.

Gibbons RJ, McDowell TL, Raman S, O'Rourke DM, Garrick D, Ayyub H, Higgs DR. 2000. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet, 24 (4), pp. 368-371. | Show Abstract | Read more

A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.

Gibbons RJ, Higgs DR. 2000. Molecular-clinical spectrum of the ATR-X syndrome. Am J Med Genet, 97 (3), pp. 204-212. | Show Abstract | Read more

Since the identification of the ATRX gene (synonyms XNP, XH2) in 1995, it has been shown to be the disease gene for numerous forms of syndromal X-linked mental retardation [X-linked alpha thalassemia/mental retardation (ATR-X) syndrome, Carpenter syndrome, Juberg-Marsidi syndrome, Smith-Fineman-Myers syndrome, X-linked mental retardation with spastic paraplegia]. An attempt is made in this article to review the clinical spectrum associated with ATRX mutations and to analyse the evidence for any genotype/phenotype correlation.

McDowell TL, Gibbons RJ, Sutherland H, O'Rourke DM, Bickmore WA, Pombo A, Turley H, Gatter K, Picketts DJ, Buckle VJ et al. 1999. Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes. Proc Natl Acad Sci U S A, 96 (24), pp. 13983-13988. | Show Abstract | Read more

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Smith ZE, Higgs DR. 1999. The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression. Hum Mol Genet, 8 (8), pp. 1373-1386. | Show Abstract | Read more

We have studied replication throughout 325 kb of the telomeric region of a human chromosome (16p13.3) and related the findings to various aspects of chromosome structure and function (DNA sequence organization, nuclease-hypersensitive sites, nuclear matrix attachment sites, patterns of methylation and gene expression). The GC-rich isochore lying adjacent to the telomere, which contains the alpha-globin locus and many widely expressed genes, replicates early in the cell cycle regardless of the pattern of gene expression. In subtelomeric DNA, replication occurs later in the cell cycle and the most telomeric region (20 kb) is late replicating. Juxtaposition of early replicating DNA next to the telomere causes it to replicate later in S-phase. Analysis of the timing of replication in chromosomes with deletions, or in transgenes containing various segments of this telomeric region, suggests that there are no critical origins or zones that initiate replication, rather the pattern of replication appears to be related to the underlying chromatin structure which may restrict or facilitate access to multiple, redundant origins. These results contrast with the pattern of replication at the human beta-globin locus and this may similarly reflect the different chromosomal environments containing these gene clusters.

Higgs DR. 1998. Do LCRs open chromatin domains? Cell, 95 (3), pp. 299-302. | Read more

Picketts DJ, Tastan AO, Higgs DR, Gibbons RJ. 1998. Comparison of the human and murine ATRX gene identifies highly conserved, functionally important domains. Mamm Genome, 9 (5), pp. 400-403. | Read more

Flint J, Bates GP, Clark K, Dorman A, Willingham D, Roe BA, Micklem G, Higgs DR, Louis EJ. 1997. Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains. Hum Mol Genet, 6 (8), pp. 1305-1313. | Show Abstract | Read more

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub-domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non-homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.

Flint J, Thomas K, Micklem G, Raynham H, Clark K, Doggett NA, King A, Higgs DR. 1997. The relationship between chromosome structure and function at a human telomeric region. Nat Genet, 15 (3), pp. 252-257. | Show Abstract | Read more

We have sequenced a contiguous 284,495-bp segment of DNA extending from the terminal (TTAGGG)n repeats of the short arm of chromosome 16, providing a full description of the transition from telomeric through subtelomeric DNA to sequences that are unique to the chromosome. To complement and extend analysis of the primary sequence, we have characterized mRNA transcripts, patterns of DNA methylation and DNase I sensitivity. Together with previous data these studies describe in detail the structural and functional organization of a human telomeric region.

HOMI J, LEVEE L, HIGGS D, THOMAS P, SERJEANT G. 1997. Pulse oximetry in a cohort study of sickle cell disease Clinical and Laboratory Haematology, 19 (1), pp. 17-22. | Read more

Gibbons RJ, Bachoo S, Picketts DJ, Aftimos S, Asenbauer B, Bergoffen J, Berry SA, Dahl N, Fryer A, Keppler K et al. 1997. Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nat Genet, 17 (2), pp. 146-148. | Read more

Picketts DJ, Higgs DR, Bachoo S, Blake DJ, Quarrell OW, Gibbons RJ. 1996. ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome. Hum Mol Genet, 5 (12), pp. 1899-1907. | Show Abstract | Read more

It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with alpha thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.

Flint J, Rochette J, Craddock CF, Dodé C, Vignes B, Horsley SW, Kearney L, Buckle VJ, Ayyub H, Higgs DR. 1996. Chromosomal stabilisation by a subtelomeric rearrangement involving two closely related Alu elements. Hum Mol Genet, 5 (8), pp. 1163-1169. | Show Abstract | Read more

We have characterised a subtelomeric rearrangement involving the short arm of chromosome 16 that gives rise to alpha-thalassaemia by deleting the major, remote regulatory element controlling alpha-globin expression. The chromosomal breakpoint lies in an Alu family repeat located only approximately 105 kb from the 16p subtelomeric region. The broken chromosome has been stabilised with a newly positioned telomere acquired by recombination between this 16p Alu element and a closely related subtelomeric Alu element of the Sx subfamily. It seems most likely that this abnormal chromosome has been rescued by the mechanism of telomere capture which may reflect a more general process by which subtelomeric sequences are normally dispersed between chromosomal ends.

Raynham H, Gibbons R, Flint J, Higgs D. 1996. The genetic basis for mental retardation. QJM, 89 (3), pp. 169-175. | Read more

Gibbons RJ, Higgs DR. 1996. The alpha-thalassemia/mental retardation syndromes. Medicine (Baltimore), 75 (2), pp. 45-52. | Show Abstract | Read more

The chromosome-16 and the X-chromosome forms of alpha-thalassemia--ATR-16 and ATR-X--exemplify 2 important causes of syndromal mental retardation. ATR-16 is a contiguous gene syndrome which arises from loss of DNA from the tip of chromosome 16p13.3 by truncation, interstitial deletion, or unbalanced translocation. It provided the first example of a chromosome translocation that could be detected by molecular analysis but not conventional cytogenetics. It also provided the first example of a telomeric truncation giving rise to a complex genetic syndrome. In contrast ATR-X appears to be due to mutations in a trans-acting factor that regulates gene expression. Mutations in transcription factors have recently been identified in a number of genetic diseases (for example, Denys-Drash syndrome, WT1 [19]; pituitary dwarfism, PIT1 [16]; Rubinstein-Taybi syndrome, CBP [20]. Not only is this mechanism proving to be an important cause of complex syndromes but it is providing new perspectives on certain developmental pathways. XH2 may not be a classical transcription factor but it is certainly involved in the regulation of gene expression, exerting its effects on several different genes. It seems likely that other mutations in this class of regulatory proteins will be found in patients with complex disorders including mental retardation. In broader terms the 2 mechanisms described here may prove to be responsible for a significant proportion of mental retardation. However, without a feature such as alpha-thalassemia to pinpoint the area of genome or pathways involved it may prove difficult to identify other, similarly affected genes underlying other forms of mental retardation. As the human genome project and rapid genome analysis evolve this problem should become less of an obstacle. In the meantime, it is very worthwhile to continue looking for unusual clinical associations that may point to critical genes underlying human genetic disorders.

Vyas P, Vickers MA, Picketts DJ, Higgs DR. 1995. Conservation of position and sequence of a novel, widely expressed gene containing the major human alpha-globin regulatory element. Genomics, 29 (3), pp. 679-689. | Show Abstract | Read more

We have determined the cDNA and genomic structure of a gene (-14 gene) that lies adjacent to the human alpha-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the alpha-globin genes lies within intron 5 of this gene. Analysis of the -14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the alpha-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the alpha-globin promoters and not the -14 gene promoter, which lies between the alpha promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the -14 gene adjacent to the alpha cluster have been maintained since the bird/mammal divergence, 270 million years ago.

Gourdon G, Sharpe JA, Higgs DR, Wood WG. 1995. The mouse alpha-globin locus regulatory element. Blood, 86 (2), pp. 766-775. | Show Abstract

We have identified and cloned the major alpha globin locus regulatory element in the mouse (m alpha RE). This element shows a high level of sequence homology to its human counterpart (HS -40) and lies between the same two exons of an upstream, widely expressed gene in both species. Footprinting and band shift studies of the core element show conservation of many (but not all) of the protein binding sites identified as functionally important in HS -40. The functional equivalence of the mouse element was shown by attaching it to a human alpha globin gene and examining expression in transgenic mice. Readily detectable levels of human alpha mRNA were produced in these mice but they were lower than the endogenous gene expression and did not show copy number dependence. These results suggest that sequences additional to this major regulatory element may be necessary to obtain complete regulation of the alpha globin genes in both species.

Craddock CF, Vyas P, Sharpe JA, Ayyub H, Wood WG, Higgs DR. 1995. Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments. EMBO J, 14 (8), pp. 1718-1726. | Show Abstract

Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions.

Gibbons RJ, Picketts DJ, Villard L, Higgs DR. 1995. Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell, 80 (6), pp. 837-845. | Show Abstract | Read more

The ATR-X syndrome is an X-linked disorder comprising severe psychomotor retardation, characteristic facial features, genital abnormalities, and alpha-thalassemia. We have shown that ATR-X results from diverse mutations of XH2, a member of a subgroup of the helicase superfamily that includes proteins involved in a wide range of cellular functions, including DNA recombination and repair (RAD16, RAD54, and ERCC6) and regulation of transcription (SW12/SNF2, MOT1, and brahma). The complex ATR-X phenotype suggests that XH2, when mutated, down-regulates expression of several genes, including the alpha-globin genes, indicating that it could be a global transcriptional regulator. In addition to its role in the ATR-X syndrome, XH2 may be a good candidate for other forms of X-linked mental retardation mapping to Xq13.

McPherson EW, Clemens MM, Gibbons RJ, Higgs DR. 1995. X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: a new kindred with severe genital anomalies and mild hematologic expression. Am J Med Genet, 55 (3), pp. 302-306. | Show Abstract | Read more

We report a new kindred containing 4 patients with X-linked alpha-thalassemia/mental retardation syndrome ((ATR-X). Like previously reported ATR-X patients, these children are all genetic males with severe developmental delay and characteristic facial appearance. The genital anomalies are more severe than in most previous cases and have led to a female sex of rearing for 3 of the 4 patients. The hematologic expression is extremely mild and was not demonstrable on routine hematologic studies including hemoglobin electrophoresis, but the three living patients all had hemoglobin H inclusions on brilliant cresyl blue stained peripheral smears. The combination of skewed X-inactivation and haplotype analysis at Xq12-q21.3 confirmed carrier status in the 3 obligate carriers in the kindred and led to identification of an additional carrier. Two other women in the kindred appear to be noncarriers on the basis of normal X-inactivation and/or inheritance of a different Xq12-21.3 haplotype. More widespread use of brilliant cresyl blue staining for HbH inclusions in individuals with the facial phenotype of ATR-X and/or ambiguous genitalia may lead to the identification of more affected patients and improved understanding of the clinical spectrum of ATR-X.

Gibbons RJ, Picketts DJ, Higgs DR. 1995. Syndromal mental retardation due to mutations in a regulator of gene expression. Hum Mol Genet, 4 Spec No (REV. ISS.), pp. 1705-1709. | Show Abstract

Mental handicap is a common clinical problem that has been a relatively neglected area of research. Though the causes are varied and complex, molecular biologists are making progress in understanding the mechanisms in some cases, particularly where there are distinguishing phenotypic or genetic markers. The fortuitous association of alpha thalassaemia with a form of mental retardation has allowed us to define a specific X-linked syndrome (ATR-X). Positional cloning was used to define a disease interval and examination of candidate genes demonstrated that mutations in a gene, XH2, showing homology to the SNF2 superfamily were responsible for this syndrome. The complex ATR-X phenotype suggests that this gene, when mutated, down-regulates the expression of several genes including the alpha-globin genes indicating that it could be a global transcriptional regulator. It is conceivable that this mechanism is involved in other forms of syndromal mental retardation.

Flint J, Craddock CF, Villegas A, Bentley DP, Williams HJ, Galanello R, Cao A, Wood WG, Ayyub H, Higgs DR. 1994. Healing of broken human chromosomes by the addition of telomeric repeats. Am J Hum Genet, 55 (3), pp. 505-512. | Show Abstract

We have characterized and compared a series of naturally occurring chromosomal truncations involving the terminal region of the short arm of human chromosome 16 (16p13.3). All six broken chromosomes appear to have been stabilized by the direct addition of telomeric repeats (TTAGGG)n to nontelomeric DNA. In five of the six chromosomes, sequence analysis shows that the three of four nucleotides preceding the point of telomere addition are complementary to and in phase with the putative RNA template of human telomerase. Otherwise we have found no common structural features around the breakpoint regions. These findings, together with previously reported in vitro data, suggest that chromosome-healing events in man can be mediated by telomerase and that a small region of complementarity to the RNA template of telomerase at the end of a broken chromosome may be sufficient to prime healing in vivo.

Logie LJ, Gibbons RJ, Higgs DR, Brown JK, Porteous ME. 1994. Alpha thalassaemia mental retardation (ATR-X): an atypical family. Arch Dis Child, 70 (5), pp. 439-440. | Show Abstract | Read more

A novel form of severe, X linked mental retardation associated with alpha thalassaemia (ATR-X syndrome) has recently been described. Two affected cousins are described, one of whom has an unusually mild haematological phenotype. HbH inclusions, which are the hallmark of this disease, were only detected in the peripheral red blood cells after repeated observations.

Sharpe JA, Wells DJ, Whitelaw E, Vyas P, Higgs DR, Wood WG. 1993. Analysis of the human alpha-globin gene cluster in transgenic mice. Proc Natl Acad Sci U S A, 90 (23), pp. 11262-11266. | Show Abstract | Read more

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

Sharpe JA, Summerhill RJ, Vyas P, Gourdon G, Higgs DR, Wood WG. 1993. Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression. Blood, 82 (5), pp. 1666-1671. | Show Abstract

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.

Vickers MA, Vyas P, Harris PC, Simmons DL, Higgs DR. 1993. Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere. Proc Natl Acad Sci U S A, 90 (8), pp. 3437-3441. | Show Abstract | Read more

We recently reported the presence of four genes lying between the human alpha-globin gene cluster and the telomere of the short arm of chromosome 16 (16p). We now report that one of these genes encodes 3-methyladenine DNA glycosylase, an enzyme important in the repair of DNA after damage by alkylating agents. The gene comprises five exons, representation of which differs in independently isolated cDNA clones. Although the gene is widely expressed, the abundance of its mRNA is considerably higher in a colon adenocarcinoma cell line (HT29) than in other cell lines that were tested. The major positive erythroid-specific regulatory element controlling alpha-globin gene expression lies equidistant between the promoters of the alpha-globin genes and the 3-methyladenine DNA glycosylase gene. Interestingly, in contrast to the alpha-globin genes, expression of the 3-methyladenine DNA glycosylase gene is not influenced by the regulatory element in the human erythroleukemia cell line K562.

Higgs DR, Wood WG. 1993. Understanding erythroid differentiation Current Biology, 3 (8), pp. 548-550. | Read more

Sharpe JA, Chan-Thomas PS, Lida J, Ayyub H, Wood WG, Higgs DR. 1992. Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice. EMBO J, 11 (12), pp. 4565-4572. | Show Abstract

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.

Vyas P, Sharpe JA, Watt P, Higgs DR, Wood WG. 1992. Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells. Blood, 80 (7), pp. 1832-1837. | Show Abstract

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

Vyas P, Vickers MA, Simmons DL, Ayyub H, Craddock CF, Higgs DR. 1992. Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin. Cell, 69 (5), pp. 781-793. | Show Abstract | Read more

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.

Gibbons RJ, Suthers GK, Wilkie AO, Buckle VJ, Higgs DR. 1992. X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis. Am J Hum Genet, 51 (5), pp. 1136-1149. | Show Abstract

We have examined seven pedigrees that include individuals with a recently described X-linked form of severe mental retardation associated with alpha-thalassemia (ATR-X syndrome). Using hematologic and molecular approaches, we have shown that intellectually normal female carriers of this syndrome may be identified by the presence of rare cells containing HbH inclusions in their peripheral blood and by an extremely skewed pattern of X inactivation seen in cells from a variety of tissues. Linkage analysis has localized the ATR-X locus to an interval of approximately 11 cM between the loci DXS106 and DXYS1X (Xq12-q21.31), with a peak LOD score of 5.4 (recombination fraction of 0) at DXS72. These findings provide the basis for genetic counseling, assessment of carrier risk, and prenatal diagnosis of the ATR-X syndrome. Furthermore, they represent an important step in developing strategies to understand how the mutant ATR-X allele causes mental handicap, dysmorphism, and down-regulation of the alpha-globin genes.

Wilkie AO, Higgs DR, Rack KA, Buckle VJ, Spurr NK, Fischel-Ghodsian N, Ceccherini I, Brown WR, Harris PC. 1991. Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell, 64 (3), pp. 595-606. | Show Abstract | Read more

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.

Thame M, Grandison Y, Mason K, Thompson M, Higgs D, Morris J, Serjeant B, Serjeant G. 1991. The red cell distribution width in sickle cell disease--is it of clinical value? Clin Lab Haematol, 13 (3), pp. 229-237. | Show Abstract | Read more

The red cell distribution width (RDW) has been studied during the clinical steady state in 1121 patients with homozygous sickle cell (SS) disease, 344 with sickle cell-haemoglobin C (SC) disease, 68 with sickle cell-beta+ thalassaemia, 49 with sickle cell beta 0 thalassaemia and in 130 control subjects with a normal (AA) genotype. The mean RDW was moderately increased in S beta + thalassaemia and SC disease and markedly increased in S beta 0 thalassaemia and SS disease. In SS, SC and S beta 0 thalassaemia genotypes, lower RDW values occurred in females and with alpha thalassaemia. The RDW correlated negatively with total haemoglobin, mean cell haemoglobin concentration, mean cell volume, and fetal haemoglobin (HbF) and positively with reticulocyte count in SS disease. A low RDW was associated with higher weight and less frequent dactylitis, painful crisis, acute chest syndrome, acute splenic sequestration, and hospital admissions. A low RDW in SS disease is consistent with a high total haemoglobin, high HbF, low reticulocyte count, alpha thalassaemia, and a more mild clinical course.

Morris J, Dunn D, Beckford M, Grandison Y, Mason K, Higgs D, Ceulaer KD, Serjeant B, Serjeant G. 1991. The haematology of homozygous sickle cell disease after the age of 40 years British Journal of Haematology, 77 (3), pp. 382-385. | Read more

Wilkie AO, Pembrey ME, Gibbons RJ, Higgs DR, Porteous ME, Burn J, Winter RM. 1991. The non-deletion type of alpha thalassaemia/mental retardation: a recognisable dysmorphic syndrome with X linked inheritance. J Med Genet, 28 (10), pp. 724. | Read more

Wilkie AO, Gibbons RJ, Higgs DR, Pembrey ME. 1991. X linked alpha thalassaemia/mental retardation: spectrum of clinical features in three related males. J Med Genet, 28 (11), pp. 738-741. | Show Abstract | Read more

We describe three males (two brothers and a cousin) who have the X linked alpha thalassaemia/mental retardation (ATR-X) syndrome. The diagnosis, originally suspected in the brothers because of similarity in dysmorphic features to previous cases, was confirmed haematologically in the surviving brother. The cousin has less typical dysmorphism and a virtually normal routine blood count, but haemoglobin H inclusions were found in his red blood cells showing that he has the same condition. This report expands the clinical phenotype of the ATR-X syndrome and emphasises that a normal blood count does not exclude the diagnosis.

Gibbons RJ, Wilkie AO, Weatherall DJ, Higgs DR. 1991. A newly defined X linked mental retardation syndrome associated with alpha thalassaemia. J Med Genet, 28 (11), pp. 729-733. | Read more

Donnai D, Clayton-Smith J, Gibbons RJ, Higgs DR. 1991. The non-deletion alpha thalassaemia/mental retardation syndrome: further support for X linkage. J Med Genet, 28 (11), pp. 742-745. | Show Abstract | Read more

It has previously been suggested that the non-deletion form of the alpha thalassaemia/mental retardation syndrome may be an X linked disorder. We describe four brothers with this syndrome in whom the diagnosis was first suspected because of their characteristic clinical features, although these varied somewhat from one sib to another. The diagnosis was confirmed in each case by showing Hb H inclusions in a proportion of their red blood cells. The identification of four similarly affected boys in this pedigree is consistent with an X linked pattern of inheritance. In support of this, very rare Hb H inclusions could be found in the red blood cells of the mother and one sister who both share some facial features with the affected boys and are presumably carriers of this disorder. This pedigree thus provides further evidence that this is an X linked syndrome and indicates the clinical and haematological variability that may exist even within a single affected family.

Hatton CS, Wilkie AO, Drysdale HC, Wood WG, Vickers MA, Sharpe J, Ayyub H, Pretorius IM, Buckle VJ, Higgs DR. 1990. Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster. Blood, 76 (1), pp. 221-227. | Show Abstract

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

Higgs DR, Wood WG, Jarman AP, Vickers MA, Wilkie AO, Lamb J, Vyas P, Bennett JP. 1990. The alpha-thalassemias. Ann N Y Acad Sci, 612 pp. 15-22.

Vyas P, Higgs DR, Weatherall DJ, Dunn D, Serjeant BE, Serjeant GR. 1988. The interaction of alpha thalassaemia and sickle cell-beta zero thalassaemia. Br J Haematol, 70 (4), pp. 449-454. | Show Abstract | Read more

The effects of alpha thalassaemia on sickle cell-beta zero thalassaemia have been studied by comparing haematological and clinical features in four subjects homozygous for alpha thalassaemia 2 (2-gene group), 27 heterozygotes (3-gene group), and 55 with a normal alpha globin gene complement (4-gene group). Alpha thalassaemia was associated with significantly higher haemoglobin levels and lower reticulocyte counts independent of the presence of splenomegaly. Contrary to expectation, alpha thalassaemia was associated with small but significant increases in mean cell volume and mean corpuscular haemoglobin concentration. Splenomegaly at age 5 years and episodes of acute splenic sequestration were significantly more frequent in the 4-gene group. There were no significant differences in painful crises, acute chest syndrome, or other clinical features.

Weatherall DJ, Bell JI, Clegg JB, Flint J, Higgs DR, Hill AV, Pasvol G, Thein SL. 1988. Genetic factors as determinants of infectious disease transmission in human communities. Philos Trans R Soc Lond B Biol Sci, 321 (1207), pp. 327-348. | Show Abstract | Read more

Genetic factors may play an important role in individual susceptibility to infection. Hitherto this problem has been investigated by attempting to relate the distribution of genetic polymorphisms in populations to present or past infection, or by analysing specific infections by classical twin studies or group comparisons. There is reasonable evidence that the common red-cell polymorphisms involving haemoglobin, enzymes or membrane have been maintained by relative resistance to malaria. Blood-group heterogeneity, including secretor status, may reflect varying susceptibility to bacterial, virus and yeast infection. There is increasing evidence that the HLA-DR system may be involved in modifying the clinical course of bacterial, virus and parasitic infection. So far no specific resistance or susceptibility loci similar to those found in murine models have been found in man. DNA analysis, particularly involving restriction fragment length polymorphism associations with candidate genes, offers a valuable new approach to this problem.

Wainscoat JS, Pilkington S, Peto TE, Bell JI, Higgs DR. 1987. Allele-specific DNA identity patterns. Hum Genet, 75 (4), pp. 384-387. | Show Abstract | Read more

A method of genetic analysis is presented which involves digestion of DNA with a single restriction enzyme (PvuII) and hybridisation with a mixture of five probes. Four of the five probes chosen recognise hypervariable regions (HVRs) of the human genome and hence an allele-specific DNA identity pattern results. An advantage of this approach to genetic characterisation is that the complex identity patterns may be broken down into simple allelic systems of known chromosomal localization by hybridisation with the individual probes. Also different probes may be included in a combined probe designed for particular types of investigation.

Vandenplas S, Higgs DR, Nicholls RD, Bester AJ, Mathew CGP. 1987. Characterization of a new α° thalassaemia defect in the South African population British Journal of Haematology, 66 (4), pp. 539-542. | Read more

Falusi AG, Esan GJF, Ayyub H, Higgs DR. 1987. Alpha-thalassaemia in Nigeria: Its interaction with sickle-cell disease European Journal of Haematology, 38 (4), pp. 370-375. | Read more

Bowden DK, Hill AV, Higgs DR, Oppenheimer SJ, Weatherall DJ, Clegg JB. 1987. Different hematologic phenotypes are associated with the leftward (-alpha 4.2) and rightward (-alpha 3.7) alpha+-thalassemia deletions. J Clin Invest, 79 (1), pp. 39-43. | Show Abstract | Read more

We have compared the phenotypes of the two common deletion forms of alpha+-thalassemia by analysis of umbilical cord blood samples from Melanesia. Homozygotes for the leftward, 4.2-kilobase, deletion (-alpha 4.2) had significantly higher levels of Hb Bart's at birth than homozygotes for the rightward, 3.7-kilobase, deletion (-alpha 3.7). Compound heterozygotes for each deletion had intermediate values. Although deletion forms of alpha 0 thalassemia were not found in this survey, nondeletion alpha-thalassemia was present at low frequency. Since the predominant rightward deletion in this population, -alpha 3.7III, entirely removes the alpha 1-gene and the 4.2-kilobase deletion deletes the alpha 2-gene, these data indicate that the alpha 2-globin gene has a higher output than the alpha 1-gene, on single alpha-gene chromosomes.

Flint J, Hill AV, Weatherall DJ, Clegg JB, Higgs DR. 1986. Alpha globin genotypes in two North European populations. Br J Haematol, 63 (4), pp. 796-797. | Read more

Higgs DR, Wainscoat JS, Flint J, Hill AV, Thein SL, Nicholls RD, Teal H, Ayyub H, Peto TE, Falusi AG. 1986. Analysis of the human alpha-globin gene cluster reveals a highly informative genetic locus. Proc Natl Acad Sci U S A, 83 (14), pp. 5165-5169. | Show Abstract | Read more

Extensive molecular studies have characterized 15 dimorphic and 2 multiallelic genetic markers within the human alpha-globin gene cluster. Analysis of these markers in 9 populations has shown that the alpha-globin locus is remarkably polymorphic and is therefore an ideal marker on chromosome 16 for the construction of a human genetic linkage map. The combined analysis of 9 polymorphic markers has established alpha-globin haplotypes that provide the means to study the molecular genetics and common mutants of this cluster. The novel association of a conventional restriction fragment length polymorphism haplotype and linked, hypervariable regions of DNA should allow a comparison of the rate of change of such markers.

Nicholls RD, Hill AV, Clegg JB, Higgs DR. 1985. Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment. Nucleic Acids Res, 13 (21), pp. 7569-7578. | Show Abstract | Read more

We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta-globin alleles from an individual with beta-thalassaemia.

Hill AV, Bowden DK, Trent RJ, Higgs DR, Oppenheimer SJ, Thein SL, Mickleson KN, Weatherall DJ, Clegg JB. 1985. Melanesians and Polynesians share a unique alpha-thalassemia mutation. Am J Hum Genet, 37 (3), pp. 571-580. | Show Abstract

Several genetic markers that provide information on population migrations and affinities have been detected by studies of proteins and cellular antigens in blood. Analysis of DNA polymorphisms promises to yield many further population markers, and we report here the distribution of a new alpha-globin gene deletion (-alpha 3.7 III) detected by a restriction enzyme mapping. This is found frequently in Melanesians and Polynesians but not in five other populations in which alpha-thalassemia is prevalent. We used restriction enzyme haplotype analysis to support a single origin for this mutation and propose that it is a useful population marker. Its geographical distribution supports a route through Island Melanesia for the colonizers of Polynesia.

Hill AV, Nicholls RD, Thein SL, Higgs DR. 1985. Recombination within the human embryonic xi-globin locus: a common xi-xi chromosome produced by gene conversion of the psi xi gene. Cell, 42 (3), pp. 809-819. | Show Abstract | Read more

The duplicated human embryonic alpha-like globin genes encode a 5' functional zeta (xi 2) gene and a highly homologous pseudogene (psi xi 1). We have identified chromosomes with a xi 2-xi 1 rather than a xi 2-psi xi 1 arrangement by genomic mapping and oligonucleotide analysis. The DNA sequence of a cloned downstream xi-like gene provides direct evidence for the conversion of a psi xi 1----xi 1 gene, by a xi 2 gene. We present data suggesting that this gene conversion, which removed the only identifiable inactivating mutation in the psi xi 1 gene, was an interchromosomal event. The xi 2-xi 1 arrangement is common in all eight populations studied representing a previously undescribed type of polymorphism between individuals. Stable mRNA transcripts from the converted gene are absent at 16-20 weeks of gestation when transcripts from the xi 2 gene are readily detectable.

Higgs DR, Hill AV, Nicholls R, Goodbourn SE, Ayyub H, Teal H, Clegg JB, Weatherall DJ. 1985. Molecular rearrangements of the human alpha-globin gene cluster. Ann N Y Acad Sci, 445 (JUN), pp. 45-56. | Read more

Higgs DR, Ayyub H, Clegg JB, Hill AV, Nicholls RD, Teal H, Wainscoat JS, Weatherall DJ. 1985. Alpha thalassaemia in British people. Br Med J (Clin Res Ed), 290 (6478), pp. 1303-1306. | Show Abstract | Read more

Although alpha thalassaemia is rare in north Europeans, it has been identified in British people with no known foreign ancestry. Twelve such patients were studied, of whom eight shared a distinctive molecular defect, which was clearly different from defects seen in subjects of Mediterranean or South East Asian origin. A rare but specific form of alpha thalassaemia is therefore present in the British population. In addition, two patients from families of mixed racial origin were encountered who had a moderately severe form of thalassaemia (HbH disease) due to the inheritance of one form of alpha thalassaemia from the British parent and another type from the foreign parent. This shows the importance of careful genetic counselling of British patients with haematological findings of thalassaemia.

Bowden DK, Hill AV, Higgs DR, Weatherall DJ, Clegg JB. 1985. Relative roles of genetic factors, dietary deficiency, and infection in anaemia in Vanuatu, South-West Pacific. Lancet, 2 (8463), pp. 1025-1028. | Show Abstract | Read more

Hypochromic anaemia is very common among the island populations of Vanuatu in the South-West Pacific. Results of a large-scale survey show that, unexpectedly, this form of anaemia is seldom due to iron deficiency or coexistent parasitic disease. Rather, it results from a previously unsuspected high incidence of alpha-thalassaemia which has been identified only by application of DNA analysis to the populations studied. These findings suggest that hypochromic anaemia in tropical or subtropical populations should not necessarily be attributed to iron deficiency; detailed studies of iron status should be carried out before major dietary changes or fortification of food with iron are implemented.

Weatherall DJ, Higgs DH, Wood WG, Clegg JB. 1984. Genetic Disorders of Human Haemoglobin as Models for Analysing Gene Regulation Philosophical Transactions of the Royal Society B: Biological Sciences, 307 (1132), pp. 247-259. | Read more

Higgs DR, Hill AV, Bowden DK, Weatherall DJ, Clegg JB. 1984. Independent recombination events between the duplicated human alpha globin genes; implications for their concerted evolution. Nucleic Acids Res, 12 (18), pp. 6965-6977. | Show Abstract | Read more

We have examined the molecular structure of the human alpha globin gene complex from individuals with a common form of alpha thalassaemia in which one of the duplicated pair of alpha genes (alpha alpha) has been deleted (-alpha 3-7). Restriction mapping and DNA sequence analysis of the mutants indicate that different -alpha 3.7 chromosomes are the result of at least three independent events. In each case the genetic crossover has occurred within a region of complete homology between the alpha 1 and alpha 2 genes. Since the -alpha chromosomes may reflect the processes of crossover fixation and gene conversion between the two genes, their structures may provide some insight into the mechanism by which the concerted evolution of the human alpha globin genes occurs.

Wainscoat JS, Bell JI, Thein SL, Higgs DR, Sarjeant GR, Peto TE, Weatherall DJ. 1983. Multiple origins of the sickle mutation: evidence from beta S globin gene cluster polymorphisms. Mol Biol Med, 1 (2), pp. 191-197. | Show Abstract

Restriction site polymorphisms of the beta globin gene cluster of 244 Jamaican beta S chromosomes have been studied. Various patterns of restriction site polymorphisms (haplotypes) both 5' and 3' to the beta gene have been found. These two groups of haplotypes were found to be non-randomized with respect to each other. This is in contrast to normal beta A chromosomes where the 5' and 3' haplotypes are randomized. These findings together with the large number (18) of different beta S haplotypes found indicate that the beta S mutation probably has multiple origins.

Higgs DR, Goodbourn SE, Lamb J, Clegg JB, Weatherall DJ, Proudfoot NJ. 1983. Alpha-thalassaemia caused by a polyadenylation signal mutation. Nature, 306 (5941), pp. 398-400. | Show Abstract | Read more

Most eukaryotic messenger RNAs have the sequence AAUAAA 11-30 nucleotides from the 3'-terminal poly(A) tract. Since this is the only significant sequence homology in the 3' non-coding region it has been suggested that it may be a recognition site for enzymes involved in polyadenylation and/or termination of polymerase II transcription. This idea is strengthened by observations on the effect of deletion mutations in or around the AATAAA sequence on polyadenylation of late simian virus 40 (SV40) mRNA; removal of this sequence prevents poly(A) addition. Naturally occurring variants of this hexanucleotide are rare and hitherto their functional significance has not been assessed. We have now identified a human alpha 2-globin gene which contains a single point mutation in this hexanucleotide (AATAAA leads to AATAAG). The paired alpha 1 gene on the same chromosome is completely inactivated by a frame-shift mutation. This unique combination has enabled the expression of the mutant alpha 2 gene to be studied in vivo where it has been found that the accumulated level of alpha 2-specific mRNA in erythroid cells is reduced. Furthermore, readthrough transcripts extending beyond the normal poly(A) addition site are detected in mRNA obtained from HeLa cells transfected with cloned DNA from the mutant alpha 2 gene, suggesting that the single nucleotide change in the AATAAA sequence is the cause of its abnormal expression.

Serjeant B, Myerscough E, Serjeant GR, Higgs DR, Moo-Penn WF. 1982. Sickle Cell-Hemoglobin D Iran: A Benign Sickle Cell Syndrome Hemoglobin, 6 (1), pp. 57-59. | Read more

Weatherall DJ, Clegg JB, Wood WG, Old JM, Higgs DR, Pressley L, Darbre PD. 1980. THE CLINICAL AND MOLECULAR HETEROGENEITY OF THE THALASSEMIA SYNDROMES Annals of the New York Academy of Sciences, 344 (1 Fourth Cooley), pp. 83-100. | Read more

Hughes JR, Roberts N, McGowan S, Hay D, Giannoulatou E, Lynch M, De Gobbi M, Taylor S, Gibbons R, Higgs DR. 2014. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat Genet, 46 (2), pp. 205-212. | Show Abstract | Read more

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.

Law MJ, Lower KM, Voon HP, Hughes JR, Garrick D, Viprakasit V, Mitson M, De Gobbi M, Marra M, Morris A et al. 2010. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell, 143 (3), pp. 367-378. | Show Abstract | Read more

ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.

Wallace HA, Marques-Kranc F, Richardson M, Luna-Crespo F, Sharpe JA, Hughes J, Wood WG, Higgs DR, Smith AJ. 2007. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell, 128 (1), pp. 197-209. | Show Abstract | Read more

We have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse alpha globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human alpha globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human alpha thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases.

De Gobbi M, Viprakasit V, Hughes JR, Fisher C, Buckle VJ, Ayyub H, Gibbons RJ, Vernimmen D, Yoshinaga Y, de Jong P et al. 2006. A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science, 312 (5777), pp. 1215-1217. | Show Abstract | Read more

We describe a pathogenetic mechanism underlying a variant form of the inherited blood disorder alpha thalassemia. Association studies of affected individuals from Melanesia localized the disease trait to the telomeric region of human chromosome 16, which includes the alpha-globin gene cluster, but no molecular defects were detected by conventional approaches. After resequencing and using a combination of chromatin immunoprecipitation and expression analysis on a tiled oligonucleotide array, we identified a gain-of-function regulatory single-nucleotide polymorphism (rSNP) in a nongenic region between the alpha-globin genes and their upstream regulatory elements. The rSNP creates a new promoterlike element that interferes with normal activation of all downstream alpha-like globin genes. Thus, our work illustrates a strategy for distinguishing between neutral and functionally important rSNPs, and it also identifies a pathogenetic mechanism that could potentially underlie other genetic diseases.

Tufarelli C, Stanley JA, Garrick D, Sharpe JA, Ayyub H, Wood WG, Higgs DR. 2003. Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat Genet, 34 (2), pp. 157-165. | Show Abstract | Read more

Nearly all human genetic disorders result from a limited repertoire of mutations in an associated gene or its regulatory elements. We recently described an individual with an inherited form of anemia (alpha-thalassemia) who has a deletion that results in a truncated, widely expressed gene (LUC7L) becoming juxtaposed to a structurally normal alpha-globin gene (HBA2). Although it retains all of its local and remote cis-regulatory elements, expression of HBA2 is silenced and its CpG island becomes completely methylated early during development. Here we show that in the affected individual, in a transgenic model and in differentiating embryonic stem cells, transcription of antisense RNA mediates silencing and methylation of the associated CpG island. These findings identify a new mechanism underlying human genetic disease.

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