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WO1992018648A1 - Test permettant d'identifier des individus presentant un risque accru de developpement d'une leucemie lymphoide ou un lymphome - Google Patents

Test permettant d'identifier des individus presentant un risque accru de developpement d'une leucemie lymphoide ou un lymphome Download PDF

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Publication number
WO1992018648A1
WO1992018648A1 PCT/US1992/002804 US9202804W WO9218648A1 WO 1992018648 A1 WO1992018648 A1 WO 1992018648A1 US 9202804 W US9202804 W US 9202804W WO 9218648 A1 WO9218648 A1 WO 9218648A1
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Prior art keywords
seq
individual
oligonucleotide
lymphoma
rearrangement
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PCT/US1992/002804
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English (en)
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Ilan R. Kirsch
Stanley Lipkowitz
Marc-Henri Stern
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The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Publication of WO1992018648A1 publication Critical patent/WO1992018648A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates, in general, to an assay.
  • the present invention relates to an assay that identifies individuals at an increased risk for the development of lymphoid leukemia or lymphoma.
  • genomic instability is a well- established means of increasing cancer risk in a given individual or in a population at large.
  • genetic diseases in which an increase in genomic instability is known to be a part of the phenotypic spectrum of affected individuals. Invariably, such individuals have an increased risk of cancer.
  • numerous environmental exposures such as radiation and a host of environmental carcinogens are known to increase the tendency for genetic material to be subject to point mutation, viral insertion or transposition, gene amplification, deletion, or translocation.
  • a commonly used measure of genomic instability is karyotypic analysis in which the appearance of an individual's chromosomes is studied in a given number of cells usually derived from that individual's skin, bone marrow, peripheral blood, amniotic fluid, or chorionic villus biopsy. This is an extremely labor intensive and tedious procedure which could never be used (as currently practiced) as a screening test. Even in particular patients selected for karyotypic analysis it is unusual to have a cytogeneticist provide full karyotypes for over 20 metaphase cells.
  • the present invention provides a screening test that accurately measures lymphocyte-specific genomic instability and by extrapolation thus identifies individuals at increased risk for the development of lymphoid malignancy. It can be performed on a small amount of peripheral blood taken from an individual (although any lymphocyte containing fluid or tissue can be used) .
  • the screening test uses a specific set of DNA primers in a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the power and utility of PCR analysis is now common knowledge.
  • the unique application of this technique that is described here provides an almost infinitely faster and more sensitive means for detecting lymphocyte-specific genomic instability.
  • the present invention shows that the amount of genomic instability seen in a lymphocyte population can serve as a marker for an individual's or population's risk for the development of common types of lymphoid malignancy.
  • the present invention has determined that patients who suffer from the disease ataxia- telangiectasia (AT) have a 70-100 fold increase of T-lymphocyte specific inversion of chromosome 7, inv(7) (pl4q35) , than normal unaffected individuals.
  • AT disease ataxia- telangiectasia
  • pl4q35 T-lymphocyte specific inversion of chromosome 7, inv(7)
  • the present invention developed, at least in part, from the results of an analysis of a non- malignant cell line carrying one such abnormality, an inversion of chromosome 7 (inv(7)), derived from a patient with ataxia-telangiectasia (AT) .
  • AT is a disease characterized by progressive cerebellar degeneration, oculocutaneous telangiectasia, variable immunodeficiency, radiation sensitivity, chromosomal instability, and a predisposition to lymphoid malignancies (Boder, E. (1985) In: Ataxia- Telangiectasia: genetics neuropathology, and immunology of a degenerative disease of childhood. R.A. Gatti and M. Swift, eds. Alan R. Liss, inc., New York, NY. pg. 1; McKinnon, P.J. (1987) Hum. Genet. 75:197).
  • inv(7) is apparently mediated by precisely the same enzymes, "recombinases", that mediate the much more common intralocus VJ rearrangements that are known to confer the ability to mount cell mediated and humoral immune response. It is clear that these same recombinases also play a critical role in the generation of "malevolent” translocations associated with malignant transformation of lymphocytes. These "malevolent” translocations occur at a much lower frequency than the "innocent” ones previously described, but they are brought to the attention of clinicians and researchers because they contribute to the clonal outgrowth of the cell in which they occur, usually recognized when a patient presents with leukemia or lymphoma.
  • Flour Industry have a significantly increased number of translocations in their blood (Garry et al. (1989) Science 246:251-255). An analysis of the data revealed that these translocations disappeared over time if fumigant exposure ceased. Furthermore, the particular abnormalities seen look a lot like those observed in the peripheral T-cell population of AT patients. An epidemiological study reported by Dr. Michael Alavanja (Alavanja et al. (1990) J. Nat. Cancer Inst. 82:840-848) has determined that this same population is at a significantly increased risk for the development of leukemia and lymphoma. The present invention, specifically, the assay for the occurrence of the inv(7) , is being used to study this population.
  • the present invention provides novel oligonucleotides and methods of using same to identify individuals at an increased risk for the development of lymphoid leukemia and/or lymphoma.
  • the invention further provides a method for identifying carcinogenic compounds.
  • the present invention relates to an oligonucleotide that is useful in identifying individuals and/or populations of individuals with an increased risk for the development of lymphoid leukemia and lymphoma comprising a DNA sequence within an immune receptor locus capable of displaying genomic instability due to interlocus rearrangement.
  • the present invention relates to a method of identifying individuals and/or populations of individuals at an increased risk for the development of lymphoid leukemia or lymphoma comprising amplifying a region of immune receptor loci that display genomic instability due to interlocus rearrangement and analyzing the amplification products for evidence of genomic instability.
  • the present invention relates to a method for identification of individuals that are homozygous or heterozygous for the autosomal recessive disease ataxia- telangiectasia and related syndromes comprising amplifying a region of immune receptor loci of an individual wherein the loci display genomic instability due to interlocus rearrangement and analyzing the amplification products for evidence of genomic instability.
  • the present invention relates to a method of identifying carcinogenic compounds comprising exposing peripheral blood or lymphocyte cells in vitro to the compounds, amplifying a region of immune receptor loci of the cells which loci display genomic instability due to interlocus rearrangement, and analyzing the amplification products for evidence of genomic instability.
  • the present invention relates to a test kit for identifying an individual at an increased risk for developing lymphoid leukemia or lymphoma comprising at least one container means having disposed therein at least one of the above-mentioned oligonucleotides.
  • Figure 1 Idiogram of normal chromosome 7 and schematic representation of the TCR- ⁇ and TCR ⁇ loci. Oligonucleotides used as PCR primers or probes are represented by arrows. Oligonucleotide sequences and their use are described in Table I.
  • FIG. 1 Southern analysis of amplified V7- J ⁇ hybrid genes from genomic PBL DNA.' Amplified products of V ⁇ -3 ⁇ l PCR reaction were hybridized to J ⁇ lc oligonucleotide probe (SEQ ID NO: 7) (panel A) . Amplified products of V ⁇ - ⁇ ⁇ 2 PCR reaction were hybridized to J02c oligonucleotide probe (SEQ ID NO: 10) (panel B) . Size markers are in base pairs. Identical results were obtained when both blots were stripped and rehybridized to a V- ⁇ oligonucleotide probe (data not shown) . Lanes 1-5 AT, lane 6 AT heterozygote, lane 7 unaffected sibling of AT patient, lane 8-10 normal (NL) individuals.
  • FIG. 3 Southern blot analysis of amplified V ⁇ -J ⁇ hybrid genes from serially diluted genomic PBL DNA from an AT patient (AT) and a normal individual (NL) . Left panels are amplification of V ⁇ - J ⁇ hybrids and right panels are amplification of V 7 - J/32 hybrids from an AT patient (top) and a normal individual (bottom) . Size markers are in base pairs. Amount of DNA (in micrograms) added per PCR reaction is shown at the top. The blots were hybridized to the V-yc oligonucleotide probe (SEQ ID NO: 3) .
  • FIG. 4 Southern blot analysis of hybrid genes amplified from cDNA of AT patients (AT) and normal individuals (NL) .
  • First strand cDNA prepared from PBL RNA and amplified with PCR primers specific for hybrid genes was hybridized to V ⁇ c oligonucleotide probe (SEQ ID NO: 3) (panel A) or stripped and rehybridized to C3c oligonucleotide probe (SEQ ID NO: 13) (panel B) .
  • Longer exposure revealed that all normal samples were positive while control cDNA prepared from the SUP-T1 cell line was negative (data not shown) .
  • First strand cDNA amplified with PCR primers specific for rearranged TCR/? was hybridized to the Sc probe (SEQ ID NO: 13) (panel C) .
  • the present invention relates to an oligonucleotide that can be used to identify an individual with an increased risk for the development of lymphoid leukemia or lymphoma.
  • the oligonucleotide comprises a DNA segment that has a sequence corresponding to that of a region in the genome of an individual wherein the region is within an immune receptor locus which is capable of displaying genomic instability due to interlocus rearrangement.
  • the oligonucleotide includes a DNA sequence corresponding to a region within 2000 base pairs of a junction of an inv(7) .
  • the oligonucleotide has sufficient homology to hybridize to a V7, J ⁇ , or CB DNA sequence.
  • the oligonucleotide primer has the sequence of bases of an oligonucleotide in Table 1.
  • the present invention relates to a method of identifying an individual at an increased risk for the development of lymphoid leukemia or lymphoma.
  • the method comprises amplifying a region of immune receptor loci capable of displaying genomic instability due to interlocus rearrangement and analyzing the amplification products for evidence of genomic instability.
  • the rearrangement is a TCR 7 V-TCR ⁇ J rearrangement.
  • the method can be used, for example, to identify an individual who has been exposed to a leukemogenic or lymphomagenic agent, for example, an environmental agent, specifically, either a pesticide or a herbicide.
  • the present invention relates to a method of identifying (specifically, pre-natal identification) an individual homozygous or heterozygous for the autosomal recessive disease ataxia-telangiectasia and related syndromes (for example, Nijmegan breakage syndrome) .
  • the method comprises amplifying a region of immune receptor loci capable of displaying genomic instability due to interlocus rearrangement and analyzing the amplification products for evidence of genomic instability. More specifically, the products are analyzed by Southern blot analysis.
  • the present invention relates to a method for identifying carcinogenic compounds.
  • the method comprises exposing peripheral blood or lymphocyte cells in vitro to a potentially carcinogenic compound, amplifying immune receptor loci of the cells wherein the loci display genomic instability due to interlocus rearrangement and analyzing the amplification products for evidence of genomic instability.
  • the present invention relates to a test kit for identifying an individual at an increased risk for developing lymphoid leukemia or lymphoma comprising at least one container means having disposed therein at least one of the above-mentioned oligonucleotides.
  • PBL Human peripheral blood mononuclear leukocytes
  • Ficoll-Hypaque Pulcoll-Hypaque
  • density gradient centrifugation Boyuro, A. (1968) Scand. J. Clin. Invest. 21(suppl 97):77.
  • AT ataxia- telangiectasia
  • Ataxia-telangiectasia a cellular and molecular link between cancer, neuropathology, and immune deficiency. (1982) B.A. Bridges, and D.G., Harnden, eds. John Wiley, Chichester, England.
  • the AT patients were between 15 and 40 y.o and the normal individuals were between 20 and 40 y.o.
  • DNA and RNA were extracted as previously described (Blin, N. and D.W. Stafford (1976) Nucleic Acid Res. 3:2303; Chirgwin, J.M. et al (1979) Biochemistry 18:5294).
  • Negative control DNA and RNA were prepared as above from the T-cell line SUP-T1 which is a cell line without an inv(7) chromosomal abnormality (Hecht, F. et al (1984) Science 226:1445) . Separate amplification reactions were performed to assay rearrangements between V 7 and JJl segments or V 7 and J 2 segments.
  • a two step nested PCR protocol was performed by a modification of the method of Saiki et al. (Saiki, R.K. et al (1985) Science 230:1350).
  • DNA (1 ⁇ g or an appropriate dilution) was diluted into a 75 ⁇ l solution containing 200 ⁇ M dGTP, 200 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dCTP (Pharmacia), 50mM KC1, lO M Tris (pH 8.3), l.5mM MgCl 2 , 0.01%(w/v) gelatin, 2.5 units Taq polymerase (Cetus, Emeryville, CA) and 10% (v/v) di ethylsulfoxide (DMSO;Sigma, St.
  • DMSO di ethylsulfoxide
  • DMSO DMSO was added to the PCR reactions because it was found to increase the yield of larger products when amplified simultaneously with smaller products.
  • the sample was overlaid with light mineral oil (Fischer Scientific, Fairlawn, N.J.).
  • To amplify the DNA the mixture was heated to 95°C for 2.5 min, then underwent 25 cycles of 0.5 min at 95°C, 0.5 min at 50°c, and 6 min at 72°C, followed by 10 min at 72°C after the last cycle.
  • RT-PCR was performed by a modification of a previously described method (Doherty, P.J. et al (1989) Anal. Biochem. 177:7).
  • First strand cDNA was synthesized in a 20 ⁇ l reaction containing 5.0 ⁇ g of unfractionated total RNA, ImM dGTP, ImM dATP, ImM dTTP, ImM dCTP, 75mM KC1, 50mM Tris(pH 8.3), lOmM DTT, 3mM MgCl .
  • RNA was incubated at 37°C for 30 min, then at 95 C C for 5 min (to inactivate the enzyme) and stored at -20°C.
  • Amplification of hybrids was performed by diluting 10 ⁇ l of first strand cDNA into 90 ⁇ l containing 200 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dCTP, 50mM KCL, lOmM Tris (pH 8.3), 1.5mM MgCl 2 , 0.01% (w/v) gelatin, 2.5 units Taq polymerase and 0.5 ⁇ g of Vb (SEQ ID NO: 2) and C ⁇ b (SEQ ID NO: 12) oligonucleotides (Figure 1; Table 1).
  • Oligonucleotide Sequence V a (SEQ ID NO: 1) TACATCCACTGGTACCTACACCAG V b (SEQ ID NO: 2) CTAGAATTCCAGGGTTGTGTTGGAATCAGGA V7C (SEQ ID NO: 3) TCTGGGGTCTATTACTGTGCCACCTGG V/3a (SEQ ID NO: 4) TCTGTGTACTGGTACCAACAG
  • AGCGGATCCAGCTCCGGTCCA CTCACCTGTGACCGTGAGCC
  • the location of the oligonucleotides is indicated on Figure 1.
  • the oligonucleotides labelled “a” were used as primers in the first PCR reaction.
  • the oligonucleotide labelled “b” were used as internal primers in the second "nested” reaction, the oligonucleotides labelled "c” were used as probes.
  • the V7 and V ⁇ oligonucleotides correspond to the coding strand.
  • the V7b primer has an EcoRl site introduced to facilitate subsequent cloning of PCR products.
  • the J/81, J / 92, and C ⁇ oligonucleotides correspond to the inverted complement of the respective coding strands.
  • the J ⁇ lb (SEQ ID NO: 6), JS2b (SEQ ID NO: 9), and CjSb (SEQ ID NO: 12) primers have a BamHl site introduced to facilitate subsequent cloning of PCR products. Analysis of PCR Products
  • the amplified samples were extracted with CHC1 3 to remove the mineral oil, and one half of each sample was analyzed by electrophoresis on a 1.5% agarose gel, Southern transfer (Southern, E.M.
  • Oligonucleotides used as probes were end labelled to a specific activity of 10 7 -10' cpm/ ⁇ g DNA with [ ⁇ - "p] dCTP (Amersham, Arlington, II) by terminal deoxynucleotidyl transferase (BRL) as described (Young, W.S. et al (1986) Neurosci. Lett. 70:198).
  • the Nytran membrane was prehybridized for one hour at 50°C in hybridization buffer (6xSSC, lOx
  • Amplified material (from genomic or RT-PCR) was digested with EcoRI and Ba Hl restriction endonucleases (BRL) and ligated into a pGEM-7Zf(+) plasmid vector (Promega, Madison, WI) . Transfected bacterial cells were screened for recombinants using oligonucleotides internal to the amplifying primers ( Figure 1; Table 1). Plasmid DNA was prepared cy alkaline lysis (Birnboim, H.C. and J. Doly (1979) Nucleic Acid Res. 7:1513) and subsequent ribonuclease A (Sigma) treatment. Plasmid DNA was sequenced using the dideoxy chain termination technique (Sanger, F.
  • Genomic titration results for normal and AT samples were compared by a two tailed Student's t-test.
  • the fraction of open reading frames in genomic and cDNA clones was compared by a Chi-Squared test.
  • J / 31 consists of 6 J segments
  • J>92 consists of 7 J segments.
  • Two separate sets of 3' oligonucleotides were chosen (one set in the intron 3' of J ⁇ l.6 and the other set in the intron 3' of J32.7) to allow amplification of rearrangements into either the J/Sl or J/32 locus respectively ( Figure 1; Table 1) .
  • the observed PCR products spanned the size range predicted for each locus, and hybridized to oligonucleotide probes internal to both the J3 oligonucleotide primers ( Figure 2) and the V 7 oligonucleotide primers (data not shown) .
  • the PCR amplified hybrid TCR genes were cloned into a pGEM-7Zf(+) plasmid vector and multiple clones from an AT patient and a normal individual were sequenced (Table II) . This analysis revealed that the interlocus recombination occurred in a site-specific fashion analogous to the intralocus recombination normally described for Ig or TCR genes (Waldmann, T.A. (1987) Adv. Immunol. 40:247).
  • V 7 regions showed variability in the exact nucleotide at which the recombination occurred, and were followed by a variable number of nucleotides that could not be assigned to either the V 7 or J ⁇ locus (so-called N-region nucleotides) .
  • the J/3 regions also showed variability in the exact nucleotide at which recombination occurred. There were no recognizable D/3 regions. All but one of the V 7 regions utilized could be assigned to either V 7 1.2, 1.3, 1.4, 1.5, or 1.8 - all of which are known functional V 7 genes (LeFranc, M.P. et al (1986)
  • V7 1.7 which is a non-functional V7 region due to a deletion with its coding sequence (LeFranc, M.P. et al (1986) Cell. 45:237).
  • V 7 and J ⁇ sequences there were occasionally base changes compared to reported sequences. These might represent either polymorphisms or PCR generated artifacts.
  • An open reading frame that would allow translation of a hybrid V 7 -J0 region was present in 8 of 16 AT genomic clones and 2 of 7 normal genomic clones.
  • clone 7 derived from a normal individual showed rearrangement between a V 7 region and the intron immediately 3* of J/32.7.
  • SEQ ID NO:18 (VT 1.4) GCCACCTGGG—CCTCCCCCC—TCCTATAATTCA (J ⁇ 1.6)
  • SEQ ID NO:19 (VT 1.3) GCCACCTGGGAC—TCTGTATAAGG—GCAATCAGCCC (J/9 1.5)
  • SEQ ID NO:20 (VT 1.2) GCCACCTGGG—TAA—CTACGAGCAG (J9 2.7)
  • SEQ ID NO:21 (VT 1.3) GCCACCTGGGAC—GAC—TCCTACGAGCAG (J/9 2.7) +
  • SEQ ID NO:23 1 (VT 1.2) GCCACCTGGGACGGG—CAACGTCCACGAC—CAG (J ⁇ 2.7)
  • SEQ ID NO:24 2 (VT 1.3) GCCACCTGGG—TCCGGCCTGGGAGT—TACGAGCAG (J/3 2.7) +
  • SEQ ID NO:28 6 (VT 1.5) GCCACCTGG—ACGGGAGGGG—GCACAGATACG (J/9 2.3) + SEQ ID NO: 29
  • SEQ ID NO: 30 1 (V 7 1.3) GCCACCTGGGACAGG — TTCC — TAATTCA (J ⁇ 1.6)
  • SEQ ID NO: 33 (VT 1.3) GCCACCTGGGACAGG — TAGCGGGAACGG — TACGAGCAG ( J ⁇ 2.7) -
  • SEQ ID NO:37 (VT 1.8) GCCACC—GAGTAAGCGGGGGGCCAAGGG—AGATACG (J/3 2.3) -
  • V-J junctions of hybrid genomic gene clones were assigned based on at least 90 bases of sequence. Sequence is shown beginning at the fifth codon (bold nucleotides) from the 3' end of the germline VT I genes. The J3 regions (J) were assigned based on their entire coding sequence. Sequence is shown ending with the fourth complete codon (bold nucleotides) from the 5' end of the germline J/3 genes. N-region nucleotides (N) represent those nucleotides that could not be assigned to either VT or J/3 segments. Open reading frames were considered positive if correct reading frame was maintained with respect to both VT and J/3. 'Multiple identical clones were found.
  • the frequency of hybrid TCR genes in the PBL of 5 AT patients and 5 apparently normal individuals was determined by PCR amplification of serial dilutions of the DNA samples. Separate titrations were performed on the DNA from each diluted sample using primers for either the J/31 cluster or the J32 cluster. A representative titration for V 7 -J31 or V 7 -J32 rearrangement is shown for one AT patient and one normal individual ( Figure 3) . It was consistently possible to dilute the DNA derived from AT patients 1-2 logs further (down to 10 "5 -10 "s ⁇ g) than normal individuals and still detect V ⁇ -J ⁇ hybrids.
  • the frequency of hybrid genes per 10' cells was calculated from the farthest dilution with a detectable PCR product for both loci of each sample, assuming single copy sensitivity for the PCR reaction and 1.5 x 10 5 cell equivalents per microgram of DNA.
  • the frequency for the two J ⁇ loci were added to give the frequency of hybrid genes.
  • the AT patients had 587 + 195 V ⁇ -J ⁇ recombinants/10 5 cells (range 133-1000) while the normal individuals had 8 + 1 recombinants/10 ! cells (range 4-10) , for an approximate 70 fold difference that was statistically significant (p ⁇ .02). There was no preferential utilization of the J / 31 or J32 loci for either normal or AT samples.
  • RNA transcripts from the hybrid TCR genes that had occurred in normal and AT PBL was assayed.
  • Expression of mRNA transcripts from the V ⁇ -J ⁇ recombinants was demonstrated by the use of RT-PCR ( Figure 4) .
  • a specific PCR product of the predicted size (approx. 300 bp) which hybridized to a labelled oligonucleotide internal to the V 7 primer ( Figure 4A) and to a labelled oligonucleotide internal to the C ⁇ primer ( Figure 4B) was seen in AT patients and normal individuals.
  • As a negative control no specific PCR product was seen in the SUP- Tl cell line (data not shown) .
  • the intensity of the signal from the AT samples was much greater than the intensity of the signal from the normal samples, consistent with a greater abundance of mRNA transcripts in the AT samples.
  • Dilution analysis of the first strand cDNA prepared from RNA of AT and normal PBL revealed approximately 10-100 times more transcripts from the AT samples.
  • Amplification of rearranged TCR0 (V ⁇ -J ⁇ -C ⁇ ) resulted in a product of equal signal intensity from all of the samples
  • the AT patient was found to have one predominant cDNA clone comprising 6 of 18 independent clones sequenced. No other AT clone was found in duplicate. The normal clones more frequently had duplicates and only 11 different clones were found among the 25 clones sequenced. Open reading frames were present in 11 of 12 different cDNA clones from the AT patient and 8 of 10 different cDNA clones from the normal individual. This predominance of cDNA clones maintaining a correct open reading frame was significantly greater than the results for the genomic clones (p ⁇ .005).
  • SEQ ID NO:41 4* (VT 1.3) GCCACCTGGGACAGG—CCCCTGTCGATG—GATACG (J/3 2.3) + SEQ ID NO:42
  • SEQ ID NO:55 (VT 1.4) GCCACCTGGGATGGG—CCTTTGGT—TGGGGCCAAC (J ⁇ 2.6) -.
  • Table III Sequence of V-J junctions of hybrid gene cDNA clones. Table legend same as in Table II. All clones sequenced were correctly spliced to a C ⁇ segment (data not shown) . 'Multiple identical clones were found.
  • EXAMPLE 4 Individuals exposed to Pesticides and/or Herbicides Two samples of blood were obtained from a patient who has been using pesticides for the past 20 years. One sample was collected early in season, while a second sample was collected late in the season. PCR analysis revealed that both samples were abnormal and contained a 70-100 fold increase of T-lymphocyte specific inversion of chromosome 7, inv(7) (pl4q35) . Karyotypically both samples were abnormal as well.
  • TITLE OF INVENTION A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma
  • CORRESPONDENCE ADDRESS A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma

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Abstract

L'invention concerne un oligonucléotide qui peut être utilisé pour identifier des individus présentant un risque accru de développement d'une leucémie lymphoïde et/ou d'un lymphome ainsi qu'un kit de test contenant les oligonucléotides en question. L'invention concerne également un procédé d'identification d'individus et/ou de populations d'individus présentant un risque accru de développement d'une leucémie lymphoïde et/ou d'un lymphome. L'invention concerne en outre un procédé d'identification d'individus ayant la maladie récessive autosomique de l'ataxie-télangiectasie et des syndrômes apparentés. L'invention concerne en outre un procédé d'identification de composés carcinogènes.
PCT/US1992/002804 1991-04-11 1992-04-10 Test permettant d'identifier des individus presentant un risque accru de developpement d'une leucemie lymphoide ou un lymphome WO1992018648A1 (fr)

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CELL, Vol. 37, issued July 1984, CACCIA et al., "The T cell receptor B chain genes are located on chromosome 6 in mice and chromosome 7 in humans", p. 1091-1099. *
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