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Biomedical subjects

S Keeney

Publications and source records attributed to S Keeney.

At least 37 records · Page 2Linked to original sources
PubMed1999

The methylenetetrahydrofolate reductase gene C677T polymorphism in patients with homozygous sickle cell disease and stroke.

Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism may cause hyperhomocysteinaemia, a recognized risk factor for stroke, in individuals with folate deficiency. Homozygous sickle cell (SS) disease is associated both with increased demands for folic acid and a tendency to develop stroke. We therefore investigated a possible role of the MTHFR C677T polymorphism in SS disease patients with stroke. Investigation of the frequency of the polymorphism in 48 patients with stroke and in 48 age-, sex- and racially-matched SS controls without stroke failed to reveal a difference between the groups (Fisher exact test, P = 0.99). Homozygosity for the MTHFR C677T polymorphism is unlikely to be a risk factor for stroke in this population with SS disease.

Adolescent↗
PubMed1999

Mutations in von Willebrand factor multimerization domains are not a common cause of classical type 1 von Willebrand disease.

Type 1 von Willebrand disease (vWD) is an autosomal dominant bleeding disorder of variable penetrance. It is characterised by a mild to moderate bleeding tendency and a quantitative deficiency of von Willebrand factor (vWF) with the full range of vWF multimers. Few mutations have been described which account for the mode of inheritance in dominant vWD type 1. We screened the vWF multimerization domains (regions D1-D3 of the vWF gene) of 12 unrelated patients with dominant vWD type 1 to investigate the hypothesis that multimerization of vWF sub-units may be inhibited or reduced by a "dominant negative" mechanism. Platelet-derived RNA was reverse transcribed and the resulting vWF cDNA amplified by the polymerase chain reaction (PCR) in a series of overlapping fragments. These were subjected to a combination of single-strand conformation polymorphism (SSCP) and heteroduplex analysis. This approach identified mobility shifts on acrylamide gels that represented 12 distinct SSCP and/or heteroduplex patterns in our patient group. DNA sequencing of the region encompassing each mobility shift showed these variants to represent previously described polymorphisms within the vWF coding sequence. Examination in all 12 patients for the previously described G3389T and T3445C mutations proved negative. The molecular pathology of classical type 1 vWD remains enigmatic, mutations having been identified in only a small minority of patients. A common mechanism underlying this disease state has still to be elucidated.

Amino Acid Substitution↗
PubMed1998

Type 2N von Willebrand disease: rapid genetic diagnosis of G2811A (R854Q), C2696T (R816W), T2701A (H817Q) and G2823T (C858F)--detection of a novel candidate type 2N mutation: C2810T (R854W).

The majority of patients with type 2N von Willebrand disease (VWD type 2N) have mutations in the region of the von Willebrand factor (VWF) gene encoding the factor VIII binding domain of VWF. Two mutations predominate among VWD type 2N patients: G2811A and C2696T, which respectively bring about the amino acid substitutions R854Q and R816W in VWF. Several other mutations have been found in VWD type 2N, including T2701A (H817Q) and G2823T (C858F). We have developed a genetic test which permits rapid screening for these four mutations in a single polymerase chain reaction (PCR). The test employs induced heteroduplex formation using two universal heteroduplex generators, one of which detects G2811A (R854Q) and G2823T (C858F), the other detects C2696T (R816W) and T2701A (H817Q). The allele frequency of the common G2811A (R854Q) mutation was investigated in the local (S. Wales) population by examination of 216 VWF genes (108 individuals) and was found to be 0.01. The heteroduplex-based test additionally detected a novel candidate type 2N mutation, C2810T (R854W) and a previously described polymorphism, G2805A (R852Q). The polymorphism showed allele frequencies of 0.92 (G nucleotide) and 0.08 (A nucleotide) in the population study.

Base Sequence↗
PubMed1997

Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.

Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.

Amino Acid Sequence↗
PubMed1997

The prothrombin gene G20210A variant: prevalence in a U.K. anticoagulant clinic population.

We have investigated the prevalence of a recently reported genetic variation in the prothrombin gene (G20210A) in patients with an objectively confirmed history of venous thrombosis, 12/219 patients (5.5%) were found to be heterozygous carriers of the 20210A allele. The incidence of the 20210A allele in a group of 164 healthy controls was 1.2% (allele frequency 0.61%, 95% CI 0.08-2.19). When patients with a known alternative hereditary risk factor for venous thrombosis (factor V Leiden mutation or deficiency of antithrombin, protein C or protein S) were excluded, the G20210A variant was found to increase the risk for venous thrombosis by approximately 5-fold (odds ratio 5.4, 95% CI 1.16-25.0). This prothrombin gene sequence variation adds further to the list of recognized genetic risk factors for thrombophilia.

Adult↗
PubMed1997

HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party.

The risk of developing factor VIII inhibitor antibodies in haemophilia A may relate both to factor VIII genotype and genes within the HLA complex known to influence immune response. We investigated a cohort of 176 patients with severe haemophilia A and with either high-level inhibitors (> 10BU/ml) or with no history of an inhibitor, stratified according to the presence or absence of the factor VIII gene intron 22 inversion. HLA DRB1, DQA1 and DQB1 polymorphisms were determined by PCR. HLA frequencies form 137 United Kingdom controls were used for comparison. HLA phenotype frequency differences, expressed as odds ratios with 95% confidence intervals were as follows: HLA-DRB*1501, DQB1*0602 and DQA1*0102 were all increased in frequency in patients with inhibitors, only DQA1*0102 reaching statistical significance (OR 2.7, 1.2-5.9). These alleles form part of an established HLA haplotype. The frequencies of HLA-DRB1*1501, DQB1*0602 and DQA1*0102 were particularly raised in patients with inhibitors and a factor VIII gene intron 22 inversion, although again only DQA1*0102 achieved significance (OR 3.1, 1.0-10.1). The frequency of DRB1*01, DQB1*0501, DQA1*0101 were also increased in inhibitor patients lacking the intron 22 inversion although this failed to achieve statistical significance. This data suggests that HLA class II profile constitutes a weak risk factor for developing inhibitor antibodies to factor VIII. This may be more pronounced in patients with an intron 22 inversion.

Chromosome Inversion↗
PubMed1996

Communication between homologous chromosomes: genetic alterations at a nuclease-hypersensitive site can alter mitotic chromatin structure at that site both in cis and in trans.

BACKGROUND: In vegetatively growing diploid strains of the yeast Saccharomyces cerevisiae, homologous chromosomes appear to be paired via multiple interstitial interactions, likely as a regular feature of the diploid lifestyle. We have previously suggested that this pairing is guided by direct physical interactions between intact DNA duplexes in nuclease-hypersensitive regions and that homology is sensed directly at the DNA level. RESULTS: As a first test of this idea we have examined the level of DNase I sensitivity at a prominent nuclease-hypersensitive site in mitotic chromatin in strains that are either homozygous or heterozygous for a pair of alleles at this site. We find that the degree of nuclease sensitivity at this site on a given (maternal or paternal) chromosome can vary depending upon whether the homologue carries the same allele or the different allele. The data are suggestive that nuclease sensitivity is higher in the former case than in the latter, as though nuclease hypersensitivity might be increased when the two alleles match as compared to when they do not. CONCLUSIONS: Formally, these observations suggest that homologous chromosomes can communicate via a mechanism that senses the status of the assayed nuclease-hypersensitive site with resultant changes in chromatin structure at that site. The observed pattern of effects is fully compatible with direct physical interactions between homologues at nuclease-hypersensitive regions, but alternative scenarios also can be envisioned. Since DNase I hypersensitive sites occur in many important regions of chromosomes, homology-dependent interactions involving such regions could potentially affect diverse processes including gene expression (e.g. transvection), chromosome organization, domain structure, and/or DNA replication patterns.

Alleles↗
PubMed1996

Expression of diverse AML1/MTG8 transcripts is a consistent feature in acute myeloid leukemia with t(8;21) irrespective of disease phase.

The (8;21) chromosomal translocation occurs in 20% of adult patients with AML M2. This translocation interrupts two genes, AML1 on chromosome 21q and MTG8 (ETO) on 8q to form a chimeric gene AML1/MTG8 on the der(8) chromosome. Recent reports have shown the presence of diverse forms of transcript for this chimeric gene. Three alternative out-of-frame transcripts have been previously demonstrated (types II, III, IV) all of which have a stop codon 3' of the runt box encoding a truncated runt polypeptide. We have characterized a novel transcript (V) which is in-frame and has a stop codon 3' to the runt box. We have examined transcript diversity in 10 AML patients with t(8;21) in remission of their disease following chemotherapy or bone marrow transplantation. Specific transcripts detected at presentation in six patients were similarly expressed during remission and at relapse in two patients; thus expression of transcript diversity was unaffected by the disease phase. Alternative transcripts were unhelpful as a marker of remission quality or predictor of relapse. The significance of these diverse transcripts in leukemogenesis remains unknown.

Base Sequence↗
PubMed1996

Cisplatin-induced alterations in the expression of the mRNAs for UV-damage recognition protein.

Enhanced DNA repair is believed to be an important mechanism of the cisplatin-resistant phenotype. UV-damage recognition protein (UV-DRP) recognizes and binds to DNA lesions and may play a role in DNA nucleotide excision repair and/or replicative bypass (which is associated with post-replication repair). Potential alternations in the expression of mRNAs for UV-DRP were analyzed in this study. Two pairs of parental and cisplatin-resistant human ovarian carcinoma cell lines were utilized. Gene expression level was assessed by northern blot hybridization. No alterations in mRNA levels for the large subunit of UV-DRP were found following cisplatin treatment, whereas mRNA levels for the small subunit of UV-DRP were induced up to 4.5-fold. The time-course and concentration-response of this induction corresponded to the previously reported increase in the UV-DRP binding activity, as measured by gel shift assay. UV-DRP binding activity in cell extracts corresponds to expression of small subunit mRNA but not to expression of large subunit mRNA. These data suggest that the small subunit may be limiting for UV-DRP activity.

Antineoplastic Agents↗
PubMed1995

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

DNA, Fungal↗
PubMed1995

Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein.

DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from HeLa cells is associated with polypeptides of M(r) 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M(r) of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from HeLa cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of p127 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution). Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12.

Amino Acid Sequence↗
PubMed1995

Development of resistance to activated protein C during pregnancy.

We measured activated protein C (APC) anticoagulant activity in 20 healthy women at 14-20, 28 and 36 weeks gestation, and at 1 d post-partum. Significant reductions in the mean APC sensitivity ratio (APC-SR) were observed at all stages of pregnancy studied compared with the mean APC-SR obtained for baseline measurements carried out at > 8 weeks post-partum. APC resistance was seen in 8/19 (42%) and in 11/20 (55%) women at 14-20 and 28 weeks gestation respectively. The development of resistance to APC may contribute to the increased risk of thrombosis during pregnancy.

Blood Coagulation↗
PubMed1994

Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.

Cells, Cultured↗
PubMed1993

Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E.

A human DNA damage binding protein implicated in the DNA excision repair disorder xeroderma pigmentosum E was purified to near homogeneity from HeLa cells. The protein is abundant (approximately 10(5) copies/cell) and has a native molecular weight of 154,000-163,000 as estimated by gel filtration and glycerol gradient sedimentation. DNA damage binding activity copurified with polypeptides of 124 and 41 kDa. Based on the native molecular weight, cosedimentation of both polypeptides with DNA damage binding activity on glycerol gradients, and a molar ratio of approximately 1:1 for the two polypeptides, it appears that p124 and p41 are subunits of a heterodimeric protein. Binding to damaged DNA was resistant to K+ concentrations approaching 1 M, but showed anion-specific sensitivity to Cl- concentrations above 0.5 M, suggesting that the majority of the binding energy is contributed by nonionic interactions. In contrast to previous reports, the DNA damage binding protein was shown to recognize cyclobutane pyrimidine dimers in addition to a nonphotoreactivable lesion(s), most likely the pyrimidine-pyrimidone (6-4) photoproduct.

Chromatography, Gel↗
PubMed1993

Comparative analysis of binding of human damaged DNA-binding protein (XPE) and Escherichia coli damage recognition protein (UvrA) to the major ultraviolet photoproducts: T[c,s]T, T[t,s]T, T[6-4]T, and T[Dewar]T.

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, the damaged DNA-binding protein (DDB), is absent from some xeroderma pigmentosum complementation group E cell strains; therefore, it has been suggested that it may be the damage recognition subunit of a human excision nuclease complex. However, the identity of the UV photoproduct bound by DDB and the role of this protein in nucleotide excision repair have been controversial. In this study, we used several synthetic DNA substrates, each of which contains one of the major UV photoproducts, and DDB purified to apparent homogeneity to quantify the specific binding of DDB to various photoproducts. For comparison, the binding of the same photoproducts by the Escherichia coli damage recognition protein UvrA, which is known to be a subunit of the E. coli excision nuclease, was also measured. UvrA and DDB each bound with high affinity to T[t,s]T, T[6-4]T, and T[Dewar]T, but only marginally discriminated between an undamaged oligomer and an oligomer with a T[c,s]T. In contrast to these similarities with regard to the binding to UV photoproducts, UvrA bound to another excision repair substrate, the psoralen-thymine monoadduct, with high specificity, whereas DDB was unable to distinguish between psoralen-adducted DNA and undamaged DNA. We conclude that DDB may play a special role in the repair of UV damage, but it cannot be the sole damage recognition subunit of human excision nuclease.

Adenosine Triphosphatases↗