Biomedical subjects
V J Buckle
Publications and source records attributed to V J Buckle.
Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).
A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
The identification of a third fragile site, FRAXF, in Xq27--q28 distal to both FRAXA and FRAXE.
FRAXA is unique amongst fragile sites in that it is intimately involved with a specific clinical phenotype, the fragile X syndrome. Whilst the majority of fragile X individuals have been found to have a characteristic mutation in the FMR1 gene, a small proportion of individuals exhibiting fragility have no such mutation. Investigation of the site of chromosome fragility in these FMR1 mutation negative, fragile X site positive individuals, has identified a second site of fragility, FRAXE. However, the presence of FRAXE has not explained all such cases. Here we describe a fragile X site positive, FMR1 mutation negative family, in which chromosome fragility is not due to the FRAXA or FRAXE but is due to a third site designated FRAXF. Using fluorescent in situ hybridisation (FISH) this site is shown to lie over 1Mb distal to FRAXA. The identification of a third fragile site in this small region of the X chromosome provides an opportunity to extend our studies of the molecular nature of chromosome fragility.
Identification of the FRAXE fragile site in two families ascertained for X linked mental retardation.
Chromosome fragility in two families not exhibiting amplification of the CGG trinucleotide associated with the fragile X site has been examined. Fluorescence in situ hybridisation with cosmid DNA from loci immediately flanking FRAXA and other distal loci have confirmed that cytogenetic fragility in these subjects is the result of expression of a new folate sensitive fragile X site, FRAXE.
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.
Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12.
Apurinic/apyrimidinic (AP) sites are pre-mutagenic DNA lesions which occur spontaneously and following exposure of cells to ionising radiation or chemical mutagens. HAP1 (Human AP endonuclease 1), the major enzyme in human cells initiating repair of AP sites, shows strong sequence homology to DNA repair enzymes from bacteria, Drosophila and other mammalian species. We have cloned the HAP1 gene and determined its complete nucleotide sequence. The site of transcription initiation has been mapped to 452 bp upstream of the ATG initiation codon in the genomic DNA. The HAP1 gene consists of five exons and is unusually small (less than 2.6 kb from transcription initiation site to polyadenylation sequence) with 54% of the protein coding region and the entire 3' untranslated region contained within a single exon. The first exon is non-coding. Regions of three exons show sequence homology to the E.coli xth (exonuclease III) gene. Using in situ hybridisation, the HAP1 gene has been localised to human chromosome 14q 11.2-12.
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.
X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.
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.
High-resolution genetic map around the spinal muscular atrophy (SMA) locus on chromosome 5.
Although autosomal recessive spinal muscular atrophy (SMA) has been mapped to chromosome 5q12-q13, there is for this region no genetic map based on highly informative markers. In this study we present the mapping of two previously reported microsatellite markers in 40 CEPH and 31 SMA pedigrees. We also describe the isolation of a new microsatellite marker at the D5S112 locus. The most likely order of markers (with recombination fractions given in parentheses) is 5cen-D5S6-(.02)-D5S125-(.04)-(JK53CA1/2,D5S11 2)-(.04)-D5S39-qter. The relative order of D5S6, D5S112, and D5S39 was confirmed by in situ hybridization. Multipoint linkage analysis in 31 SMA families indicates that the SMA locus lies in the 6-cM interval between D5S6 and JK53CA1/2, D5S112.
Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion.
A high proportion of patients with myelodysplasia show characteristic karyotypic abnormalities in bone marrow cells. The most distinctive of the myelodysplastic syndromes is the 5q- syndrome characterized by refractory anemia, poorly lobulated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 (5q deletion) as the sole karyotypic abnormality. Recently, several genes encoding hemopoietic growth factors and receptors, comprising the interleukins 3, 4, and 5, macrophage colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, and the receptor for macrophage-colony-stimulating factor [the CSF1R (formerly FMS) gene product], have been localized to the long arm of chromosome 5, and there has been much speculation that deletion of one or more of these genes may be critical to the pathogenesis of the associated myeloid disorders. One candidate gene is CSF1R, which is required for normal proliferation and differentiation of hemopoietic cells of the myeloid lineage. We have carried out a molecular examination of the CSF1R, both on the 5q- chromosome and on the apparently normal homologous chromosome 5, in 10 patients with myelodysplasia and a 5q deletion. We have found, using restriction fragment length polymorphism analysis and gene dosage experiments, that all 10 patients showed deletion of CSF1R; 6 of 10 were hemizygous and 4 of 10 homozygous for CSF1R loss. The homozygous CSF1R loss has been confirmed in 2 patients by an in situ hybridization technique comparing the signal in affected cells to that in control sex-mismatched cells on the same slides. In those patients considered to have homozygous CSF1R loss by DNA experiments the gene was deleted from the 5q chromosome in all cells and from the apparently normal chromosome 5 in a subset of cells. This loss of one CSF1R allele, together with loss in some cells of the remaining allele on the homologous chromosome 5, in patients with myelodysplasia indicates that this is a region of critical gene loss on 5q. The loss of the hemopoietic growth factor receptor gene CSF1R may be important in the pathogenesis of human myeloid leukemia.
Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16.
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.
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.
Clonality of cell populations in refractory anaemia using combined approach of gene loss and X-linked restriction fragment length polymorphism-methylation analyses.
We have used X-linked restriction fragment length polymorphism (RFLP)-methylation and gene deletion analyses to investigate the nature of the progenitor cell of origin in the myelodysplastic syndromes (MDS). Gene deletion studies were performed on the granulocyte and T-lymphocyte fractions of six women with refractory anaemia (RA) and either a partial deletion of the long arm of chromosome 5 (5q-) or monosomy 7. All six showed gene loss in the granulocyte but not the T-lymphocyte fractions, indicating monoclonality of the granulocytes but not the T-lymphocytes. In order to further investigate this finding, we subsequently performed X-RFLP-methylation studies using the probe M27 beta, and also a probe for the phosphoglycerate kinase (PGK) gene. These studies have confirmed the monoclonality of the granulocytes and the polyclonality of the T-lymphocytes in these cases. Our findings suggest that in this group of patients with MDS the T-lymphocytes were not involved in the disorder, and furthermore, in the one case where B-lymphocytes were also available, that the progenitor cell of origin was restricted to the myeloid lineage.
Structure and polymorphism of human telomere-associated DNA.
We have analyzed the DNA sequences associated with four different human telomeres. Two are members of distinct repeated sequence families which are located mainly but not exclusively at telomeres. Two are unique in the genome, one deriving from the long arm telomere of chromosome 7 and the other from the pseudoautosomal telomere. One telomere-associated repeated sequence has a polymorphic distribution among the chromosome ends, being present at a different combination of ends in different individuals. These data thus identify a new source of human genetic variation and indicate that the canonical features of the organization of telomere-associated DNA are widely conserved in evolution.
Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster.
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.
Structure of the granulocyte macrophage colony-stimulating factor gene in patients with the myelodysplastic syndromes.
DNA samples from 76 patients with the myelodysplastic syndromes, including 10 cases with a partial deletion of the long arm of chromosome 5 (5q-), were examined for structural rearrangements of the granulocyte/macrophage colony-stimulating factor (GM-CSF) gene. No abnormalities were detected, indicating that structural aberrations of this gene are not a feature of the myelodysplastic syndromes.
Localisation of a dystrophin-related autosomal gene to 6q24 in man, and to mouse chromosome 10 in the region of the dystrophia muscularis (dy) locus.
We have localised a dystrophin-related autosomal gene called DMDL (Duchenne muscular dystrophy-like) to human chromosome 61q24 by in situ hybridisation. Using restriction fragment length polymorphism analysis in two mouse species, we have localised the homologous gene Dmdl in the mouse to chromosome 10 proximal to the Myb oncogene. A neuromuscular disease locus dystrophia muscularis (dy) has previously been assigned to this region of mouse chromosome 10.
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.