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D R Higgs

Publications and source records attributed to D R Higgs.

At least 19 recordsLinked to original sources
PubMed2001

Mutations in the general transcription factor TFIIH result in beta-thalassaemia in individuals with trichothiodystrophy.

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.

Cells, Cultured↗
PubMed2001

Identification of a conserved erythroid specific domain of histone acetylation across the alpha-globin gene cluster.

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.

Acetylation↗
PubMed2001

The human GPI1 gene is required for efficient glycosylphosphatidylinositol biosynthesis.

The first step in glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis that is defective in paroxysmal nocturnal haemoglobinuria is mediated by an N-acetylglucosaminyl transferase expressed in the endoplasmic reticulum. Six human genes encode subunits of this enzyme, namely PIG-A, PIG-C, PIG-H, PIG-P, GPI1, and DPM2. Here, the human GPI1 gene is characterised. This gene is organised into eleven exons. The locus was mapped to chromosome 16p13.3 near the haemoglobin alpha chain locus. GPI1 is expressed ubiquitously in human cells and tissues. Expression levels are markedly elevated in haematopoietic tissues (bone marrow, foetal liver). To determine whether human GPI1 is essential for human GPI biosynthesis, antisense RNA was expressed in HEK293 cells. Transfectants exhibited a marked but incomplete decrease in the expression of a GPI-linked reporter protein, confirming that GPI1 is required for efficient GPI biosynthesis. In contrast, expression of GPI-linked proteins is normal in lymphatic cell lines from individuals with the alpha thalassaemia/mental retardation syndrome, which is characterised by large deletions from chromosome 16p removing one of the two GPI1 alleles along with the haemoglobin alpha locus. In conclusion, GPI1 plays an important role in the biosynthesis of GPI intermediates. Due to its autosomal localisation, the heterozygous deletion of GPI1 does not lead to an overt defect in the expression of GPI-linked proteins.

Cell Line, Transformed↗
PubMed2001

Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16.

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.

Adolescent↗
PubMed2001

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

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.

Animals↗
PubMed2001

Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain.

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.

Alternative Splicing↗
PubMed2001

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Cell Nucleus↗
PubMed2001

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

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.

Adolescent↗
PubMed2000

alpha-thalassemia resulting from a negative chromosomal position effect.

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.

Chromosome Mapping↗
PubMed2000

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Alternative Splicing↗
PubMed2000

Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia.

Alpha-thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex-polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of alpha-thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)

Base Sequence↗
PubMed2000

Molecular-clinical spectrum of the ATR-X syndrome.

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.

Abnormalities, Multiple↗
PubMed2000

Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.

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.

Blotting, Southern↗
PubMed2000

A nonsense mutation of the ATRX gene causing mild mental retardation and epilepsy.

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.

Adolescent↗
PubMed1999

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

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.

Animals↗
PubMed1999

The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression.

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.

Animals↗