WO1991010912A1 - Etude de sensibilite en cas de maladie hereditaire - Google Patents

Etude de sensibilite en cas de maladie hereditaire Download PDF

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Publication number
WO1991010912A1
WO1991010912A1 PCT/GB1991/000024 GB9100024W WO9110912A1 WO 1991010912 A1 WO1991010912 A1 WO 1991010912A1 GB 9100024 W GB9100024 W GB 9100024W WO 9110912 A1 WO9110912 A1 WO 9110912A1
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WIPO (PCT)
Prior art keywords
drp
phosphate
extracts
glucosamine
cell
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PCT/GB1991/000024
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English (en)
Inventor
Tomas Lindahl
Lisa R. Karam
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Imperial Cancer Research Technology Limited
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Publication of WO1991010912A1 publication Critical patent/WO1991010912A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • the present invention relates to assays for inherited conditions.
  • AT telangiectasia
  • AT cells differ from normal cells in that they are less able to slow down replicative DNA synthesis after exposure to ionizing radiation.
  • Four different genetic complementation groups, A/B, C, D, and E, have been identified and the group A/B gene has been localized to chromosome 11 q22-23.
  • SUBSTITUTESHEET The DNA chain breaks which result from exposure to ionizing radiation, and drugs such as bleomycin and neocarzinostatin differ from those generated by nucleases. This is because abstraction of an intra-molecular H atom from a deoxyribose moiety generates a sugar radical which causes the formation of either a strand break or an oxidized apyri idinic site (Karam et al 1988). Such strand interruptions have complex and heterogeneous end group structures and would require short-patch excision-repair prior to re-ligation. For example, in one mode of cleavage, a 3' glycolate terminus is a reaction product. Another type of DNA strand break has a 3'-phosphate and an oxidized 5' terminal residue with a deoxyribonucleoside aldehyde moiety.
  • SUBSTITUTE SHEET One aspect of the present invention provides a method of determining whether a biological sample is associated with ataxia telangiectasia (AT), comprising the step of analysing the sample for the presence of glucosamine-6-phosphate or for the presence of a compound indicative of glucosamine-6-phosphate or for glucosamine-6-phosphate-for ing activity.
  • AT ataxia telangiectasia
  • a second aspect provides a method of determining whether a biological sample is associated with AT, compromising the step of analysing the sample for the presence of inhibitory factor Y, as defined herein, or for a compound indicative of Y.
  • glucosamine-6-phosphate is found in homozygous AT cells and, to a lesser extent, in heterozygous cells but is substantially absent in normal (non-AT) cells. Its lack in normal cells appears to be attributable to the presence of inhibitory factor Y.
  • Glucosamine-6-phosphate elutes from an ion-exchange HPLC column as described in more detail below to give a peak substantially corresponding to the peak labelled dRp-X in Figure 1.
  • radiolabelled (32p) dRp may be exposed to an AT cell extract to generate ( **, *- , P) glucosamine-6-phosphate so that a labelled peak can be detected.
  • SUBSTITUTESHEET Glucosamine-6-phosphate is generated from dRp in three concert enzymatic steps, partly along the so-called pentose phospha pathway, involving reaction intermediates as follows:-
  • fructose-6-phosphate > glucosamine-6-phosphate
  • a cofactor in the middle step is seduheptulose-7-phosphate.
  • AT cells are mutated such that feedback inhibition of of the three enzymes involved in the steps above (probably of the first two) is lost, leading to an increased level glucosamine-6-phosphate.
  • Increased levels of glyceraldehyde phosphate and/or fructose-6-phosphate may also be associa with AT and these compounds would therefore be examples compounds "indicative of glucosamine-6-phosphate” .
  • Increased levels we mean a level which is not found in non cells but which is associated with AT cells.
  • inhibitory compound Y is that compou obtainable from non-AT cells, which suppresses the formation glucosamine-6-phosphate by extracts of AT cells. Inhibit compound Y appears to be dialyzable and heat-stable.
  • the biological sample which is assayed may be a cell extr for example the supernatant following cell disruption spinning down at 10 000 xg, from any convenient cell of body, for example cheek cells obtained from a mouthw Alternatively, glucosamine-6-phosphate, compound Y, a comp indicative of either, or glucosamine-6-phosphate activity ma detected in body fluids such as blood, serum, saliva or urin
  • glucosamine-6-phosphate or of Y compounds such as metabolites or precursors, which, by t presence, indicate the presence of glucosamine-6-phosphate o respectively in either the sample being analysed or in the c of the patient from whom the sample is deri
  • Dephosphorylated glucosamine-6-phosphate, ie glucosamine ma suitable for this purpose.
  • the compound Y may be novel and forms a further aspect of invention.
  • the assays of the invention are useful in identifying homozygotes and heterozygotes, at least as far as AT groups C and E are concerned.
  • AT heterozygotes represent only about 1% of Caucasian population, it is thought that they may represent of those suffering from breast cancer. Clinicians would expose patients who are diagnosed as being AT homozygotes
  • SUBSTITUTESHEET heterozygotes to diagnostic X-rays, therapeutic X-rays or ga rays, or other mutagens and such patients would be warned avoid exposure to mutagens, particularly tobacco smoke.
  • homozygotes and heterozygotes would be advised not to work the nuclear industry or in radiology departments and so Conversely, non-AT homozygotes might be able to benefit f higher doses of therapeutic X-rays or gamma rays or chemi mutagens than are currently considered to be safe for population as a whole.
  • Patients may be screened as part of general "lifesty screening and counseling, as well as prior to speci diagnostic procedures or treatment.
  • Prenatal testi especially for AT homozygotes may be carried out on samples foetal tissue or amniotic fluid. Families with AT genes can genetically counselled.
  • Figure 1 shows chromatographic traces following separation anion-exchange HPLC of radioactive products following incubat of [ 32 P]2-deoxyribose-5-phosphate (dRp) with extracts of
  • Figure 2 shows chromatographic traces following separation anion-exchange HPLC of radioactive products following incuba of [ 32 P]dRp with AT (GM03189A) crude extracts (A), dial extracts (B) , boiled extracts (C), and a mixture of dialyzed boiled extracts (D).
  • Each 100 ⁇ l reaction mixture contained nmol [ 32 P] dRP (10,000 cpm).
  • Conditions for extract treat and incubation are described in Materials and Metho Reference compounds are as indicated in Fig. 1;
  • Figure 3 shows respective total ion gas chromatograms authentic glucosamine-6-phosphate (A) and purified dRp-X ( and
  • Figure 4 shows respective mass spectra of peak 2 in each Figures 3A and 3B.
  • Lymphoblastoid cell lines representative of various inherit disorders were grown in suspension culture at 37°C in RP medium 1640 supplemented with 15% foetal bovine serum a antibiotics.
  • Ataxia telangiectasia cell lines GM02782 GM01526B, GM09581, GM03332A, GM03189A were obtained from Human Genetic Mutant Cell Repository (Camden, N.J. , U.S.A.) strains B6796 and C2610 from Dr A.M. Taylor (Paterson Instit for Cancer Research, Birmingham, U.K.).
  • Cell lines GM01526 B6796 and C2610 are representative of genetic complementat groups E, D and C, respectively (A.M. Taylor, perso communication) .
  • the assay measures the modification of [ 32 P]dRp by extracts.
  • Reaction mixtures 100 ⁇ l contained 50 mM Hepes (pH 8.0), 5 mM dithiothreitol, 1 mM NH C1, 0.1 mM EDTA, 400 ⁇ g extract protein and 5 ⁇ M [ 32 P]dRp (5,000 - 20,000 cpm).
  • mixture was incubated at 37°C for 120 min and the reac stopped y heating for 5 min at 100°C, cooling and agita with lOO ⁇ l chloroform/ isoamyl alcohol (24:1, v/v) .
  • a centrifugation the aqueous phase was injected directly ont
  • the reference compounds were 2-deoxyribose-5-phosphate (Sigma which was detected by the diphenylamine reaction (Burton, 1956 [ 32 P]dRp, and 32 P0 4 3 ⁇ .
  • [ 3 P]dRp was prepared by depurinati of [ 32 P]dGMP (Amersham) in 0.5 M HC1 for 16 hr at 20°C, follow by neutralization with NaOH and -removal of free guanine a remaining traces of dGMP by adsorption to Norit charcoa Reduction of the carbonyl of the dR moiety of dPr and dRp-X the corresponding alcohol group was performed by incubation wi 0.3 M sodium borohydride at 25°C for 30 min (Goffin et al 1987). Reduction was monitored by the increased retention ti during HPLC separation.
  • the ⁇ -elimination derivative of dRp w prepared as previously described (Franklin and Lindahl, 1988).
  • Example 1 Modification of 2-deoxyribose-5-phosphate by at telanqiectasia cell extracts.
  • the dRp-X product was purified by HPLC and treated by h (65°C, 30 min or 100°C, 10 min) or acid (0.5 M HC1, 60 min), rechromatographed in the same position, suggesting that a sta covalent adduct of dRp had been formed. Similarly to unmodif dRp, this compound did not adsorb to activated charcoal. dRp-X did not co-chromatograph with deoxymononucleotides, deoxyribose-1-phosphate, a reduced form of dRp, or a sug phosphate containing a 2'-3' double bond obtained by rele from DNA by ⁇ -elimination.
  • radioactively labelled dRp-X was stron suppressed (>60%) by addition of 0.1 mM non-radioactive dRp deoxyribose-1-phosphate to reaction mixtures; partial inhibit (20-50%) was observed with a 10-fold higher concentration several other compounds including deoxyribose, ribose, ribos
  • Example 2 Cell line distribution of dRp modification
  • dRp-X yield was measured as the abi of cell extracts to generate modified dRp (dRp-X activity) .
  • dRp-X activity was lost on dialysis or heating of AT extra but could be regenerated by mixing boiled and dialyzed extracts (Fig. 2). This suggests that at least two compon are required for dRp-X production:
  • a heat-stable, low molecular weight component which does adsorb to activated charcoal and resists acid or al treatment (0.2 M HC1 or NaOH, lh at 20°C) . From various ass we conclude that this co-factor is not a divalent cation, ATP, or a free base (adenine, guanine, uracil, cytosine, thymine) .
  • AT cell line Normal cell line dRp yield GM03189A GM01953A (% of total radioactivity)
  • Each 100 ⁇ l reaction mixture contained 0.5 nmole [ 3 P]dRp an either 20 ⁇ l of a single cell or 40 ⁇ l of a 1:1 (v/v) cel extract mixture.
  • the dRp-X yield was quantified as the ratio o radioactive material in the dRp-X peak vs. total radioactiv material in the assay.
  • the dRp-X was identified as glucosamine-6-phosphate by t layer chromatography for 90 min on plates of PEI-cellul (obtained from Merck), initially in a solvent system of 500 pyridine formate (pH 4.5), and then in ten other solv systems.
  • the migration of dRp-X was identical to that authentic glucosamine-6-phosphate (obtained from Sigma) in systems.

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Abstract

Les sujets homozygotes ou hétérozygotes atteints d'ataxie-télangiectasies (AT) peuvent être détectés par l'analyse d'extraits cellulaires ou de liquides organiques à la recherche de dRp-X (un composé modifié de phosphate et de désoxyribose, identifié sous le nom de glucosamine-6-phosphate), d'activités de formation du dRp-X ou d'un composé inhibiteur Y qui est présent dans les cellules normales et inhibe la formation de dRp-X. Les homozygotes et hétérozygotes atteints d'AT sont anormalement sensibles aux mutagènes tels que la bléomycine, la fumée du tabac et les radiations ionisantes. Cette étude de sensibilité doit donc être effectuée avant l'administration médicale de certains médicaments ou rayons X et faire partie des conseils prodigués au patient sur son mode de vie.
PCT/GB1991/000024 1990-01-09 1991-01-09 Etude de sensibilite en cas de maladie hereditaire WO1991010912A1 (fr)

Applications Claiming Priority (2)

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GB9000476.3 1990-01-09
GB909000476A GB9000476D0 (en) 1990-01-09 1990-01-09 Assay for inherited condition

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Journal of Biotechnology, vol. 1, no. (5-6), December 1984, Elsevier Science Publishers B.V., (Amsterdam, NL), M. Shaham et al.: "Further characterization of the ataxia-telangiectasia clastogenic factor by reversed-phase liquid chromatography", pages 351-354 *
Mutation Research, vol. 236, no. 1, July 1990, Elsevier Science Publishers B.V., (Amsterdam, NL), L.R. Karam et al.: "Modification of deoxyribose-phosphate residues by extracts of ataxia telangiectasia cells", pages 19-25 *
The Biochemical Journal, vol. 188, no. 3, 15 June 1980, The Biochemical Society, (London, GB), M.J. Edwards et al.: "An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of gamma-irradiation-induced DNA damage", pages 677-682 *

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