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Douglas R Higgs

Publications and source records attributed to Douglas R Higgs.

At least 19 recordsLinked to original sources
PubMed2007

Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence.

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.

Animals↗
PubMed2006

A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter.

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.

Binding Sites↗
PubMed2006

Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.

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.

Alleles↗
PubMed2006

A novel mutation in the last exon of ATRX in a patient with alpha-thalassemia myelodysplastic syndrome.

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.

Aged↗
PubMed2006

Coregulated human globin genes are frequently in spatial proximity when active.

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.

Animals↗
PubMed2006

How transcriptional and epigenetic programmes are played out on an individual mammalian gene cluster during lineage commitment and differentiation.

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.

Animals↗
PubMed2005

Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences.

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.

Animals↗
PubMed2005

SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data.

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.

Algorithms↗
PubMed2005

Somatic point mutations in RUNX1/CBFA2/AML1 are common in high-risk myelodysplastic syndrome, but not in myelofibrosis with myeloid metaplasia.

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.

Base Sequence↗
PubMed2005

The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis.

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.

Animals↗
PubMed2004

Acquired alpha-thalassemia in association with myelodysplastic syndrome and other hematologic malignancies.

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.

Hematologic Neoplasms↗
PubMed2004

Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2.

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.

Acetylation↗
PubMed2004

A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain.

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.

Amino Acid Sequence↗
PubMed2004

Evaluation of alpha hemoglobin stabilizing protein (AHSP) as a genetic modifier in patients with beta thalassemia.

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.

Adolescent↗
PubMed2004

Co-inheritance of Hb Pak Num Po, a novel alpha1 gene mutation, and alpha0 thalassemia associated with transfusion-dependent Hb H disease.

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.

Amino Acid Sequence↗
PubMed2004

Comparative analysis of the alpha-like globin clusters in mouse, rat, and human chromosomes indicates a mechanism underlying breaks in conserved synteny.

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.

Animals↗
PubMed2004

Gene regulation in hematopoiesis: new lessons from thalassemia.

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.

Animals↗