The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation.

A major challenge for human genetics is to identify new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in more than half of all cases. We have developed a strategy to screen for the abnormal inheritance of subtelomeric DNA polymorphisms in individuals with mental retardation and have detected three abnormalities in 99 patients with normal routine karyotypes. Pulsed-field gel electrophoresis and reverse chromosome painting showed that one case arose from an interstitial or terminal deletion and two from the de novo inheritance of derivative translocation chromosomes. At least 6% of unexplained mental retardation is accounted for by these relatively small chromosomal abnormalities, which will be an important resource in the characterization of the genetic basis of neurodevelopment.

Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.

Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

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

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.

Chromosomal localization of GABAA receptor subunit genes: relationship to human genetic disease.

Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a beta subunit and three isoforms of the alpha subunit. The alpha 2 and beta genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The alpha 1 gene is on chromosome 5 (bands q34-q35) and the alpha 3 gene is on the X chromosome. The alpha 3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human alpha 3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression.

New methods in cytogenetics.

Developments in the technique of fluorescence in situ hybridization (FISH) now permit hybridization of sequences ranging from 1 kb to whole genomes. The technique can be used in applications from coarse mapping of whole chromosomes to high-resolution analysis of extended strands of DNA. The complexity, and hence the coverage, of 'paints' prepared by amplification is being improved to the extent that such methods are used in cloning strategies for the generation of region-specific probes. Interphase analysis and comparative genomic hybridization are becoming important tools in cancer cytogenetics, and the potential for routine analysis of fetal cells obtained from maternal blood may provide a fresh approach to prenatal cytogenetic screening. Functional studies of gene activity and nuclear organization are now also possible.

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome.

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.

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.

Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).

The hallmarks of the X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome are severe psychomotor retardation, minor facial anomalies, genital abnormalities, and an unusual form of alpha-thalassemia. The demonstration of HbH inclusions in red blood cells after incubation with brilliant cresyl blue confirms the diagnosis. We describe 15 previously unreported cases and analyse the phenotypic and hematologic findings in these subjects and compare them with previously published cases. This study demonstrates the consistency of the main characteristics of this syndrome and extends the phenotype. Developmental changes in phenotype, in particular the coarsening of the facial appearance, are illustrated. The hematologic findings are shown to vary widely; in some cases the manifestation of alpha-thalassemia may be subtle and missed without repeated examination.

Acute promyelocytic transformation in a case of acute myelomonocytic leukemia.

An unusual case of acute myelomonocytic leukemia, with marked bone marrow eosinophilia and basophilia, is described in which there was cytogenetic evidence at presentation of acute promyelocytic transformation, with acquisition of the standard 15;17 translocation.

Translocation 1;7 in hematologic disorders--a report of three further cases. Absence of amplification of the gene for the epidermal growth factor receptor.

We report three new cases with a hematologic disorder and the unbalanced translocation der(1)t(1;7)(p11;p11). It has been speculated that the gene for the epidermal growth factor receptor, localized to the short arm of chromosome 7, might be amplified in cases with this translocation. We have demonstrated that there is no amplification of this gene in these three cases.

Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells.

Transmissible chromosomal instability, characterized by non-clonal cytogenetic aberrations with a high frequency of chromatid-type aberrations together with a lower frequency of chromosome-type aberrations, has been demonstrated in the clonal descendants of human haemopoietic stem cells after alpha- but not X-irradiation. Comparable cytogenetic abnormalities have also been demonstrated in non-clonal cultures of alpha-irradiated primary human bone marrow, but a different pattern of delayed aberrations, mainly of chromosome-type, was found after X-irradiation in non-clonal cultures. In clonal analyses, delayed apoptotic cell death was evident after both X- and alpha-irradiation. It is suggested that the type of radiation exposure, the type of cell and its genetically determined susceptibility are factors that may influence the expression of delayed effects of radiation.

Cytogenetic heterogeneity of translocations associated with Duchenne muscular dystrophy.

Lymphoblastoid cell lines have been established from nine female patients with Duchenne muscular dystrophy who had previously been reported to have chromosome translocations with breakpoints in the Xp21 region. A detailed cytogenetic comparison of prometaphase chromosomes in these cell lines revealed that six of the translocations had X chromosome breakpoints in the sub-band Xp212 and that one further breakpoint could be assigned to either Xp212 or Xp213. These findings confirm and extend previous observations and provide strong evidence for Xp212 as the site of the Duchenne and Becker loci. For the remaining two translocations the simplest explanation for the observed banding pattern is that the X chromosome breakpoint lies a few thousand kilobases away, in the sub-band Xp211. Other explanations which assume breaks in Xp212 combined with complex local chromosome rearrangements are also presented. It is also possible that the altered banding pattern in these two cases is due to the influence of local sequences on the staining or uncoiling properties of the chromatin.

Localization of the horse (Equus caballus) alpha-globin gene complex to chromosome 13 by fluorescence in situ hybridization.

The alpha-globin gene complex in Equus caballus has been mapped by fluorescence in situ hybridization to the telomeric region of the long arm of chromosome 13. This is the first equine gene to be mapped to this chromosome.

Localisation of a sequence, 7C22, showing close linkage to the cystic fibrosis locus.

The DNA sequence 7C22 is known to show close linkage to the cystic fibrosis locus on chromosome 7. We present a regional localisation for this sequence by in situ hybridisation to 7q31.1----q31.2.

Localization of the human tyrosine hydroxylase gene to 11p15: gene duplication and evolution of metabolic pathways.

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH) are consecutive enzymes in the metabolic pathway leading to the production of catecholamine neurotransmitters. A comparison of recently available sequence data of these enzymes in the rat indicates about 70% homology in the 3' coding regions. We have localized TH by in situ hybridization to human chromosome region 11p15. Consideration of this assignment and that of PAH to chromosome 12, together with the known distribution of other pairs of related genes on these two chromosomes, provides convincing evidence of their ancestral relationship and suggests a role for gene duplication in the diversification of metabolic pathways in the vertebrate ancestors of mammals.

Localization of the human dopamine beta hydroxylase (DBH) gene to chromosome 9q34.

A human cDNA clone for dopamine beta hydroxylase (DBH) has been isolated from a phaeochromocytoma library. In situ hybridization of this probe to replication-banded chromosomes has localized the gene to chromosome 9q34. The structural gene for the enzyme is therefore close to the ABO blood group locus. This suggests that the previously described activity variation in levels of serum DBH may reflect alterations in either the structure or regulation of the DBH coding sequences. Both biochemical and genetic evidence therefore indicate independence of DBH from the pterin-dependent aromatic amino acid hydroxylases of the neurotransmitter pathways.

Localisation of human alpha globin to 16p13.3----pter.

A female child with alpha thalassaemia trait, moderate mental retardation, and dysmorphic features has inherited an abnormal chromosome 16 complement as a result of the unbalanced segregation of a maternal balanced translocation. Cytogenetic analysis indicates that the patient is monosomic for 16p13.3----pter and trisomic for 10q26.13----qter. DNA studies show that the patient has not inherited either maternal alpha globin allele. This accounts for the alpha thalassaemia trait in the child and places the human alpha globin complex in band 16p13.3----pter.

Telomere-related sequences at interstitial sites in the human genome.

The ends (telomeres) of eukaryotic chromosomes are protected from degradation and from loss during DNA replication by buffers of simple tandem repetitive sequence. The nucleotide sequence of these telomeric arrays is fundamental to telomere function as a site for protein and ribonucleoprotein binding and varies only slightly in a wide range of organisms. We present evidence that arrays of this human telomeric sequence, TTAGGG, are present not only at the ends of human chromosomes but also at numerous interstitial sites. These interstitial loci share nucleotide sequence similarity outside the repetitive array, suggesting that they are related functionally or have evolved from a common progenitor locus.