WO1992018648A1 - A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma - Google Patents
A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma Download PDFInfo
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- 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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Classifications
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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
The present invention relates to an oligonucleotide that can be used to identify individuals with an increased risk for the development of lymphoid leukemia and/or lymphoma and to a test kit containing the oligonucleotides. The invention also relates to method of identifying individuals and/or populations of individuals at an increased risk for the development of lymphoid leukemia and/or lymphoma. The invention further relates to a method for identification of individuals for the autosomal recessive disease ataxia-telangiectasia and related syndromes. The invention further relates to a method of identifying carcinogenic compounds.
Description
A SCREENING TEST THAT IDENTIFIES INDIVIDUALS AT
INCREASED RISK FOR THE DEVELOPMENT OF LYMPHOID
LEUKEMIA AND LYMPHOMA
BACKGROUND OF THE INVENTION Field of the Invention
The present invention relates, in general, to an assay. In particular, the present invention relates to an assay that identifies individuals at an increased risk for the development of lymphoid leukemia or lymphoma.
Background Information The induction of genomic instability is a well- established means of increasing cancer risk in a given individual or in a population at large. There are certain 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. Similarly, 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) . 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. We all carry such presumably "innocent" or innocuous cell-type specific chromosomal abnormalities in our peripheral blood T-cells, of which the inv(7) is one example. But in normal individuals all such cell- type specific chromosomal aberrations are rare, occurring at a frequency between 0.1 and 0.01% of the peripheral T-cell population. These "innocent" cell-type specific chromosomal aberrations that we all carry are caused by site-specific recombination between two of our immune receptor loci (im unoglobulin or T-cell receptor) . Recombination
that occurs between loci of the immunoglobulin (Ig) ' and T-cell receptor (TCR) genes was previously described by molecular characterization of an inversion of chromosome 14 in a T-cell lymphoma cell line (Baer, R. et al (1985) Cell 43:705; Denny, C.T. et al (1986) Nature (Lond) 320:549; Baer, R. et al
(1987) Cell 50:97). The inversion resulted from site-specific recombination between an Ig heavy chain variable (V) region and a TCR7 joining (J) region. This recombination resulted in an in-frame, transcribed hybrid gene that could be translated into a functional hybrid antigen receptor. Denny, C.T. et al ((1986) Science 234:197) described a second example of a hybrid Ig-TCR gene in the leukemic cells from a patient with acute lymphocytic leukemia. Again an in-frame hybrid gene was formed that was transcribed and could potentially be translated.
These two hybrid genes came from malignant lymphocytes. However, the contribution of these hybrid genes to the growth of the cells in which they were found is unclear since morphologically identical chromosomal abnormalities occur in peripheral blood lymphocytes (PBL) from normal individuals without any sign or symptom of lymphoid malignancy (Aurias, A. et al (1985) Hum. Genet. 71:19; Hecht, F. et al (1987) Cytogenet. 26:95). In fact, as stated previously, a variety of inversions and translocations involving two sites on both chromosome 7 and 14 can be seen in the metaphase chromosomes from normal lymphocytes (Aurias, A. et al (1985) Hum. Genet. 71:19; Hecht, F. et al (1987) Cancer Genet. Cytogenet. 26:95; Welch, J.P. et al (1975) Nature. 255:241). The breakpoints of these abnormalities coincide with the locations of the TCR and Ig heavy chain loci (Human Gene Mapping 10 (1989) Cytogenet. Cell Genet. 51) .
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). Between 1 and 5% of the peripheral T-lymphocyte metaphases in this patient, from whom the cell line was derived, carried an inv(7) but there was no evidence of a lymphoid malignancy (Stern, M.H. et al (1989) Blood 73:1285). It was determined that the inv(7) in the cell line resulted from site-specific recombination between a V region of TCR-7 and a J region of TCR (Stern, M.H. et al (1989) Blood 74:2076). Again the hybrid gene was in-frame, transcribed, and could potentially be translated into a hybrid antigen receptor. This hybrid gene could also be found in the PBL from the patient.
The formation of the 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. Thus, the process that generates innocent and malevolent translocations in lymphocytes is largely the same, but the frequency and consequences of their occurrence is quite different. Patients with AT have an increased frequency of "innocent" translocations and an analogously (in the same order of magnitude) increased tendency to develop lymphoid malignancy compared with matched non-AT populations. (Studies that form the basis of the present invention reveal that an increased frequency of formation of innocent lymphocyte-specific chromosomal abnormalities can serve as a marker for an increased risk for the development of the malevolent forms. The present invention provides a screening test for use in a normal population to diagnose an increased risk of lymphoma. For example, Dr. Vincent Garry of the University of Minnesota has reported that fumigant applicators in the U.S. 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.
SUMMARY OF THE INVENTION It is a general object of this invention to identify individuals at an increased risk for lymphoid leukemia or lymphoma.
It is a specific object of this invention to provide a method of identifying individuals at an increased risk for lymphoid leukemia or lymphoma. It is a specific object of this invention to provide an oligonucleotide for use in identifying individuals with an increased risk for the development of lymphoid leukemia or lymphoma. It is a further specific object of this invention to provide a method for use in identification of individuals homozygous or heterozygous for the autosomal recessive disease ataxia-telangiectasia and related syndromes. It is another object of the invention to provide a method for identifying carcinogenic compounds after in vitro exposure of peripheral blood or lymphocyte subpopulations to such compounds. It is a further object of the invention to provide a test kit for identifying individuals at an increased risk for lymphoid leukemia or lymphoma.
Further objects and advantages of the present invention will be clear from the description that follows.
In one embodiment, 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.
In another embodiment, 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.
In a further embodiment, 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.
In yet another embodiment, 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.
In a further embodiment, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Idiogram of normal chromosome 7 and schematic representation of the TCR-γ and TCRϊ loci. Oligonucleotides used as PCR primers or probes are represented by arrows. Oligonucleotide sequences and their use are described in Table I.
Figure 2. Southern analysis of amplified V7- Jβ hybrid genes from genomic PBL DNA.' Amplified products of Vη-3βl PCR reaction were hybridized to Jβlc oligonucleotide probe (SEQ ID NO: 7) (panel A) . Amplified products of Vη-~β2 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-γ 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.
Figure 3. Southern blot analysis of amplified Vη-Jβ hybrid genes from serially diluted genomic PBL DNA from an AT patient (AT) and a normal individual (NL) . Left panels are amplification of Vη- Jβ hybrids and right panels are amplification of V7- 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) . The apparent paradoxical increased yield of larger products at lower DNA concentrations resulted from the inefficient amplification of large products in the presence of competing small products (i.e., in upper left panel) . Increased yield of the larger products resulted when the DNA was diluted to a point that the small product was no longer present. Longer exposure of the autoradiograph revealed faint bands corresponding to the larger product in the first five lanes (data not shown) .
Figure 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) .
DETAILED DESCRIPTION OF THE INVENTION
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. In one preferred embodiment, the
oligonucleotide includes a DNA sequence corresponding to a region within 2000 base pairs of a junction of an inv(7) . In a further preferred embodiment, the oligonucleotide has sufficient homology to hybridize to a V7, Jβ, or CB DNA sequence. In yet another preferred embodiment, the oligonucleotide primer has the sequence of bases of an oligonucleotide in Table 1.
In another embodiment, the present invention relates to a method of identifying an individual at an increased risk for the development of lymphoid leukemia or lymphoma. Broadly, 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. In one preferred embodiment, the rearrangement is a TCR7V-TCRβJ 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.
In another embodiment, 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) . Broadly, 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. In another embodiment, the present invention relates to a method for identifying carcinogenic compounds. Broadly, 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.
In a further embodiment, 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.
The invention is described in further detail in the following non-limiting Examples.
EXAMPLES
The following protocols and experimental details are referenced in the examples that follow: Poly erase Chain Reaction (PCR) on DNA and RNA
Human peripheral blood mononuclear leukocytes (PBL) were obtained by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) density gradient centrifugation (Boyuro, A. (1968) Scand. J. Clin. Invest. 21(suppl 97):77.) from heparinized blood from patients with a clinically established diagnosis of ataxia- telangiectasia (AT) (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.) or from healthy normal subjects. 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 V7 and JJl segments or V7 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). In the first step, DNA (1 μg or an appropriate dilution) was diluted into a 75 μl solution containing 200μM dGTP, 200μM dATP, 200μM dTTP, 200μM dCTP (Pharmacia), 50mM KC1, lO M Tris (pH 8.3), l.5mM MgCl2, 0.01%(w/v) gelatin, 2.5 units Taq polymerase (Cetus, Emeryville, CA) and 10% (v/v) di ethylsulfoxide (DMSO;Sigma, St. Louis, MO) , and 0.1 μg of each of the primers to amplify V7-J/31 rearrangements (V7a (SEQ ID NO: 1) and ~βla. (SEQ ID N0:5)) (Figure 1; Table I) or 0.1 μg of each of the primers to amplify V'l-~β2 rearrangements (V7a (SEQ ID NO: 1) and J02a (SEQ ID NO: 8)) (Figure 1; Table I). Oligonucleotides were synthesized on an Applied Biosystems 380B (Foster City, CA) DNA synthesizer and used without further purification. 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. In the second step, 5 μl of the first amplification reaction was diluted into 70 μl of an identical solution containing either 0.5 μg of each of the nested primers to amplify the Vη-Jβ rearrangements (Vηrb (SEQ ID NO: 2) and Jølb (SEQ ID
NO: 6)) or 0.5 μg of each of the nested primers to amplify the V7-J02 rearrangement (Vb (SEQ ID NO: 2) and J£2b (SEQ ID NO: 9)) (Figure 1; Table 1). An identical thermal protocol was then performed. A consistent finding was that at high DNA concentrations, when multiple competing targets were present, short products amplified more efficiently than larger products. At lower DNA concentrations, when the larger product titrated further than the smaller product, the amplification of the larger product increased - despite fewer templates initially (e.g. Figure 3) . Longer exposures of the autoradiograph demonstrated the presence of the larger amplified products as faint bands when the shorter products were present.
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 μl reaction containing 5.0 μg of unfractionated total RNA, ImM dGTP, ImM dATP, ImM dTTP, ImM dCTP, 75mM KC1, 50mM Tris(pH 8.3), lOmM DTT, 3mM MgCl. (BRL 5X buffer; BRL, Gaithersburg, MD) , 200 units of M-MLV reverse transcriptase (BRL) , and 0.4 μg of the C a oligonucleotide primer (SEQ ID NO: 11) (Figure 1; Table 1). Equal amounts of RNA, measured by O.D. at 260nM and by visual assessment on an ethidium bromide stained agarose gel, were used for each reaction. The reaction mix was incubated at 37°C for 30 min, then at 95CC for 5 min (to inactivate the enzyme) and stored at -20°C.
Amplification of hybrids was performed by diluting 10 μl of first strand cDNA into 90 μl containing 200μM dATP, 200μM dTTP, 200μM dCTP, 50mM KCL, lOmM Tris (pH 8.3), 1.5mM MgCl2, 0.01% (w/v) gelatin, 2.5 units Taq polymerase and 0.5μg of Vb (SEQ ID NO: 2) and Cβb (SEQ ID NO: 12) oligonucleotides (Figure 1; Table 1). The sample was heated to 95°C for 2.5 min,
then underwent 50 cycles of 0.5 min at 95°C, 0.5 min at 55°C and 2 min at 72°C followed by 10 min at 72°C after the last cycle. Control amplification of intact TCRβ mRNA was performed by diluting 2 μl of first strand cDNA to 10 μl with water and then diluting this into 90 μl of PCR mixture as described above, except containing 0.5 μg of the Vβa oligonucleotide (SEQ ID NO: 4) (Figure 1; Table 1) instead of the V7b oligonucleotide (SEQ ID NO: 2) . The same thermal profile was performed.
Table I. Sequence of oligonucleotides used as PCR primers or probes.
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
5) TTCCCAGCAACTGATCATTG 6) CCAGGATCCCCCGAGTCAAGA 7) CATACCTGTCACAGTGAGCC 8) TTGCAGAGCTGACCC
9) AGCGGATCCAGCTCCGGTCCA 10) CTCACCTGTGACCGTGAGCC
11) CAGCTCAGCTCCACGTGGTC 12) GAAGGATCCTGTGGCCAGGCACACCAGTGT
13) TGTGGGAGATCTCTGCTTCTGAT
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 Vβ 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 Cβ oligonucleotides correspond to the inverted complement of the respective coding strands. The Jβlb (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 CHC13 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.
(1975) J. Mol. Biol. 98:503) to a Nytran membrane (Schleicher and Schuell, Keene, N.H.) and hybridization to appropriate oligonucleotide probes internal to the amplification primes (Figure 1; Table 1). Alkaline transfer (Reed, K.C., and D.A. Mann. (1985) Nucleic Acids Res. 13:7207) was found to be inefficient at transferring fragments smaller than 500 bp and so Southern transfer was used. Oligonucleotides used as probes were end labelled to a specific activity of 107-10' cpm/μg DNA with [α- "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
Denhardt's, and 50 μg/ml sheared herring sperm DNA). Labelled oligonucleotide in 1 μg denatured sheared herring sperm DNA and fresh hybridization buffer (at 10*cp /ml) was added to the Nytran membrane and incubated overnight at 50°C. Membranes were washed three times at room temperature with 6xSSC, 0.1%(w/v) SDS followed by high temperature washes with 6xSSC, 0.1%(w/v) SDS at Tm-5°C for each oligonucleotide probe. Autoradiography was performed at -80°C with an intensifying screen and XRP film for 1-16 hours.
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. et al (1977) Proc. Natl. Acad. Sci. USA 74:5463) and Sequenase version 2.0 (United States Biochemical Corp., Cleveland, OH). Sequences were compared to published sequences for TCRJ (Toyonaga, B. et al (1985) Proc. Natl. Acad. Sci. USA 82:8624) or TCR7 (LeFranc, M.P. et al (1986) Cell 45:237; Quartermous, T. et al (1987) J. Immunol. 138:2687)
Statistics: 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.
EXAMPLE 1 The Occurrence of TCR-tV-TCRgJ Hybrids in Lymphocyte DNA from Normal Individuals and AT Patients
With the hybrid TCR gene found in the AT cell line as a precedent an analysis of such hybrid genes in the peripheral blood T-cell population of normal individuals and five other AT patients was undertaken. Interlocus recombination between TCR7 V regions and TCR3 J regions in the genomic DNA from PBL of AT patients and normal individuals was assayed by a two step PCR reaction with nested sets of oligonucleotide primers. 5' oligonucleotides were chosen that correspond to conserved sequences within the second exon of the V7I variable regions (Figure 1; Table 1) - a highly homologous family of variable regions that represent 9 of the 14 known V7 variable regions (LeFranc, M.P. et al (1986) Cell 45:237). There are two distinct clusters of J
segments within the TCR0 locus (Toyonaga, B. et al (1985) Proc. Natl. Acad. Sci. USA 82:8624). One, J/31, consists of 6 J segments, and the other, J>92, consists of 7 J segments. Two separate sets of 3' oligonucleotides were chosen (one set in the intron 3' of Jβl.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) . Each DNA sample underwent amplification with the 5' V7 oligonucleotides and the 3' J31 or 3' J32 oligonucleotides in two separate reactions (Figure 1; Table 1) . PCR products of different size are generated in this system depending on which Jβ segment is utilized from either the JJl or J/32 clusters. The predicted sizes would range between approximately 230 bp and 2350 bp for the 6 J31 segments, and would range between approximately 250 bp and 1350 bp for the 7 J/32 segments. Specific PCR products were demonstrated by agarose gel electrophoresis, Southern transfer to Nytran membranes, and hybridization to α-MP labelled oligonucleotides internal to the amplification primers (Figure 1; Table 1),
V7-J3 interlocus recombination utilizing both J31 and J32 regions was demonstrated in the genomic DNA from PBL of AT patients and normal individuals (Figure 2) . Multiple PCR products of different sizes were seen in the five AT patients, while a more limited number of PCR products of different sizes were seen in the three normal individuals
(Figure 2) . In addition, the signal intensity of PCR products from the AT patients was greater than the intensity of the PCR products in the normal individuals. An obligate heterozygote for AT and an unaffected sibling of an AT patient gave results similar to the normal individuals (Figure 2). No specific PCR products were seen in DNA from SUP-T1,
a T-cell line without cytogenetic evidence of an inversion of chromosome 7 (data not shown) . Longer exposure of the autoradiographs demonstrated faint bands corresponding to the larger predicted PCR products in all of the AT samples and some of the normal samples for the J/31 locus (data not shown) . 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 V7 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). The V7 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 V7 or Jβ 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 V7 regions utilized could be assigned to either V7 1.2, 1.3, 1.4, 1.5, or 1.8 - all of which are known functional V7 genes (LeFranc, M.P. et al (1986)
Cell. 45:237). One clone from the normal PBL (clone 6) utilized 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). Within the V7 and Jβ 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 V7-J0 region was present in 8 of 16 AT genomic clones and 2 of 7 normal genomic clones. The greater number of different sized PCR products amplified from each locus in the AT PBL DNA compared to normal PBL DNA (Figure 2) suggested a greater heterogeneity of hybrid genes in AT. This was confirmed by the sequence analysis (Table II) since no duplicate clones were found in the 16 clones fully sequenced. This observation was extended further by comparing partial sequences of 10 additional clones, which again demonstrated no duplicates. The same results were obtained in another AT patient. Similarly the more restricted heterogeneity of hybrid genes in PBL from a normal individual (Figure 2) was confirmed by sequence analysis (Table II) since duplicate clones were often found and only 9 unique clones were found in the 18 clones sequenced. Similar results were obtained in a second normal individual.
One clone (clone 7) derived from a normal individual showed rearrangement between a V7 region and the intron immediately 3* of J/32.7.
Table II. Sequences of V-J Junctions of Genomic Clones
V — N — J Open Reading Frame
AT clones
1 (V7 1.2) GCCACCTGGGACGGG--G—GAAAAA (J/9 1.4)
SEQ ID NO: 14
2 (VT 1.4) GCCACCTGGGATGG--ACCAAGCAATG—GCCC (J/3 1.5)
SEQ ID NO:15
3 (VT 1.5) GCCACCTGGGACAGG—CCGGTATA—AATTCA (Jβ 1.6)
SEQ ID NO:16 (V7 1.4) GCCACCTGGGATG—AGAATAGAGGTGG—TCAGCCC (Jβ 1.5) +
SEQ ID NO: 17 (V7 1.4) GCCACCTGGGATGGG—CCC—TCCTATAATTCA (Jβ 1.6) +
SEQ ID NO:18 (VT 1.4) GCCACCTGGG—CCTCCCCCC—TCCTATAATTCA (Jβ 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:22 0 (VT 1.2) GCCACCTGGGACG—AGATC--TCCTACGAGCAG (J/3 2.7) +
SEQ ID NO:23 1 (VT 1.2) GCCACCTGGGACGGG—CAACGTCCACGAC—CAG (Jβ 2.7)
SEQ ID NO:24 2 (VT 1.3) GCCACCTGGG—TCCGGCCTGGGAGT—TACGAGCAG (J/3 2.7) +
SEQ ID NO:25 3 (VT 1.5) GCCACCTGGGACA—CCCCATAGACCCC—CTGGGGCCAAC(J/3 2.6)-
SEQ ID NO:26 4 (VT 1.4) GCCACCTGGGATG—AC— CTGGGGCCAAC (J/9 2.6) +
SEQ ID NO:27 5 (VT 1.8) GCC—CCGCGCGAGTTC—CAAGAGACCCAG (J/9 2.5) +
SEQ ID NO:28 6 (VT 1.5) GCCACCTGG—ACGGGAGGGG—GCACAGATACG (J/9 2.3) +
SEQ ID NO: 29
Normal clones
1 (V7 1.4) GCCACCTGGGATGGG — CGTATCGATACTCCCCCTA— ATAATTCA( J/3 1.6) +
SEQ ID NO: 30 1 (V7 1.3) GCCACCTGGGACAGG — TTCC — TAATTCA (Jβ 1.6)
SEQ ID NO: 31 (VT 1.8) GCCACCTGGGA — GAGTTC — ATGAAAAA (Jβ 1.4)
SEQ ID NO: 32 ' (VT 1.8) GCCACCTGGGA — GAAGGGGGT — AGCAATCAGCCC (Jβ 1.5)
SEQ ID NO: 33 (VT 1.3) GCCACCTGGGACAGG — TAGCGGGAACGG — TACGAGCAG ( Jβ 2.7) -
SEQ ID NO: 34 * (V 1.7) GCCACCTGGGACAGG — CCCCAGCCGGGCG — CCGGGGAG(J/S 2.2) (NA)"
SEQ ID NO: 35 (VT 1.8) GCCACCTGGGA — ATG — CTTCCAGCCCCT (2%Jβ 2.7) (NA)C
SEQ ID NO:36 (VT 1.4) GCCACCTGGCATG—T—CAAAAACATT (J/3 2.4) +
SEQ ID NO:37 (VT 1.8) GCCACC—GAGTAAGCGGGGGGCCAAGGG—AGATACG (J/3 2.3) -
SEQ ID NO:38
Table II: Sequence of V-J junctions of hybrid genomic gene clones. VT variable regions (V) 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.
"This clone utilized V7 1.7 a non-functional V7 I gene due to a deletion in the coding region. Therefore determination of open reading frame is not applicable (NA) . cThis clone rearranges into the intron 3' of Jβ 2.7. Therefore determination of open reading frame is not applicable (NA) .
EXAMPLE 2 The Frequency of Hybrid Gene Formation
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 V7-J31 or V7-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 μg) than normal individuals and still detect Vη-Jβ 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 105 cell equivalents per microgram of DNA. The frequency for the two Jβ loci were added to give the frequency of hybrid genes. The AT patients had 587 + 195 Vη-Jβ recombinants/105 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.
Different sized PCR products (representing utilization of different Jβ sequences) titrated out to different dilutions - indicating that the frequency of some Vη-Jβ recombinants was greater than others. The predominant J/3 recombination varied from patient to patient.
EXAMPLE 3 Identification of TCRnrV-TCRgJ Hybrid mRNA in Normal and AT Lymphocytes
Expression of RNA transcripts from the hybrid TCR genes that had occurred in normal and AT PBL was assayed. Expression of mRNA transcripts from the Vη-Jβ 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 V7 primer (Figure 4A) and to a labelled oligonucleotide internal to the Cβ 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 (Vβ-Jβ-Cβ) , resulted in a product of equal signal intensity from all of the samples
(Figure 4C) , demonstrating that the differences seen above (Figure 4A & 4B) were not due to qualitative differences in the RNA.
The cDNA products from one AT patient and one normal individual were cloned into a pGEM-7Zf(+) plasmid vector and multiple clones were sequenced (Table III) . Sequence analysis confirmed that these clones were Vη-Jβ recombinants except for four unusual clones discussed below. Nine of 13 AT cDNA clones and 7 of 11 normal cDNA clones utilized the same V7 region (V7 1.4). This preferential V7 utilization had not been seen in the genomic clones (Table II) . This analysis also demonstrated correct RNA splicing between the V-J exons and Cβ exons in all of the clones sequenced. 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).
Two cDNA clones from the normal individual (clones 3 and 9) showed recombination between a V7 and J7 segment but this product was then spliced to a C/31 exon. These clones maintained a correct open reading frame at the V-J junction and had apparently correct RNA splicing between the V7-J7 exon and the C/31 exon. Two other cDNA clones, one from the AT patient (clone 3) and one from the normal individual (clone 7) had no J region. Instead a VT region followed by several N-region nucleotides was joined directly to the first base of the C32 exon. Both clones maintained an open reading frame at the V7- N-C/3 junction.
Table III. Sequences of V-J Junctions of cDNA clones V — N — J Open Reading Frame
AT clones
1 (VT 1.4) GCCACCTGGGATGGG—AG—TCAGCCC (Jβ 1.5) +
SEQ ID NO:39
2 (VT 1.2) GCCACCTGGGACGG—CGTCGGGGACCTCGACGGTAA—CTAC (J/3 1.2) +
SEQ ID NO:40
3 (VT 1.4) GCCACCTGGGATG—AA—GAGGACCTGAAA (Cβ 2) NAb
SEQ ID NO:41 4* (VT 1.3) GCCACCTGGGACAGG—CCCCTGTCGATG—GATACG (J/3 2.3) + SEQ ID NO:42
5 (VT 1.4) GCCACCTGGGAT—TCA—TCCTACGAGCAG (Jβ 2.7) +
SEQ ID NO:43
6 (Vη 1.5) GCCACCTGGGA—AAGGGATCTCG—GCACAGATACG (Jβ 2.3) +
SEQ ID NO:44
7 (VT 1.2) GCCACCTGG—TCCCAGGGGTGGATAA—TACGAGCAG (Jβ 2.7) -
SEQ ID NO:45
8 (VT 1.4) GCCACCTGGGATG—T—TGAG (J/S 2.1) +
SEQ ID NO:46
9 (VT 1.4) GCCACCTGGGATGG—ATTTTCT—AGCACAGATACG (Jβ 2.3) +
SEQ ID NO:47
10 (VT 1.4) GCCACCTGGG—TGAT—CTACGAGCAG (Jβ 2.7) +
SEQ ID NO:48
11 (VT 1.4) GCCACCTGGGATAGG—CG—TAATTCA (J/3 1.6) +
SEQ ID NO:49
12 (Vη 1.4) GCCACCTGGGACAGG—GTGAGC—TCCTACGAGCAG (J/3 2.7) +
SEQ ID NO:50
13 (V7 1.4) GCCACCTGGG—TACTA—CAAGAGACCCAG (J/3 2.5) +
SEQ ID NO:51 Normal clones 1 (VT 1.3) GCCACCTGGGAC—CGCCCCA—GAAAAA (J/3 1.4)
SEQ ID NO:52 2' (Vη 1.3) GCCACCTGGGAC—CTCGCCAGG—GGCTAC (Jβ 1.2) +
SEQ ID NO:53 3' (V7 1.4) GCCACC—CCTAGG—AGTAGTGATTGG (J7 2.1) +c
SEQ ID NO:54
" (VT 1.8) GCCACCTGGGATGGG—AAC—TCTGGAAACACC (Jβ 1.3) +
SEQ ID NO:55 (VT 1.4) GCCACCTGGGATGGG—CCTTTGGT—TGGGGCCAAC (Jβ 2.6) -.
SEQ ID NO:56 (VT 1.4) GCCACCTGGGATGGG—CGGAG—TGGAAACACC (Jβ 1.3) -t
SEQ ID NO:57 (VT 1.4) GCCACC—AGTTGG—GAGGACCTGAAA (Cβ 2) NAb
SEQ ID NO:58 (V7 1.4) GCCACCTGGGATGGG—A—AAGAGACCCAO (Jβ 2.5) +
SEQ ID NO:59 (V7 1.4) GCCACCTGGGATGGG—CGTGG—TGATTGG (J7 2.1) +
SEQ ID NO:60 (V 1.3) GCCACCTGGGATGG CTGAAGCT (J/3 1.1)'
SEQ ID NO:61 (V7 1.4) GCCACCT—TGG—AAGAGACCCAG (J/S 2.5) +
SEQ ID NO:62
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 Cβ segment (data not shown) . 'Multiple identical clones were found.
"No J region was identified and so open reading frame is not applicable (NA) . These are discussed further in the text. cThese clones recombined VT and JT segments which were then spliced to C/91. These are discussed further in the text.
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.
Two samples were also collected from a second individual, who had been using herbicides. Samples were collected early and late in the season. PCR analysis revealed that although the sample taken early in the season was normal, the sample taken late in the season was abnormal and contained a 70- 100 fold increase of T-lymphocyte specific inversion of chromosome 7, inv(7) (pl4q35) . Similarly, karyotypic analysis revealed that the sample taken early was normal, while the sample taken late in the season was abnormal.
* * * * * * All publications mentioned hereinabove are hereby incorporated in their entirety by reference. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Kirsch, Ilan R.
Lipkowitz, Stanley Stern, Marc-Henri
(ii) TITLE OF INVENTION: A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma (iii) NUMBER OF SEQUENCES: 62 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CUSHMAN, DARBY ' CUSHMAN
(B) STREET: 1615 L. Street, N.W.
(C) CITY: Washington
(D) STATE: D.C. (E) COUNTRY: USA
(F) ZIP: 20036-5601 (V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Word Perfect 5.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Scott, Watson T.
(B) REGISTRATION NUMBER: 26,581
(C) REFERENCE/DOCKET NUMBER: WTS/5683/82598/SRL (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 861-3000
(B) TELEFAX: (202) 822-0944
(C) TELEX: 248453 CUSH
(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TACATCCACT GGTACCTACA CCAG 24
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CTAGAATTCC AGGGTTGTGT TGGAATCAGG A 31
(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCTGGGGTCT ATTACTGTGC CACCTGG 27
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TCTGTGTACT GGTACCAACA G 21
(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TTCCCAGCAA CTGATCATTG 20
(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCAGGATCCC CCGAGTCAAG A 21
(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CATACCTGTC ACAGTGAGCC 20
(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TTGCAGAGCT GACCC 15
(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGCGGATCCA GCTCCGGTCC A 21
(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CTCACCTGTG ACCGTGAGCC 20
(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CAGCTCAGCT CCACGTGGTC 20
(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GAAGGATCCT GTGGCCAGGC ACACCAGTGT 30
(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TGTGGGAGAT CTCTGCTTCT GAT 23
(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GCCACCTGGG ACGGGGGAAA AA 22
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCCACCTGGG ATGGACCAAG CAATGGCCC 29
(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCCACCTGGG ACAGGCCGGT ATAAATTCA 29
(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCCACCTGGG ATGAGAATAG AGGTGGTCAG CCC 33
(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GCCACCTGGG ATGGGCCCTC CTATAATTCA 30
(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCCACCTGGG CCTCCCCCCT CCTATAATTC A 31
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GCCACCTGGG ACTCTGTATA AGGGCAATCA GCCC 34
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GCCACCTGGG TAACTACGAG CAG 23
(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GCCACCTGGG ACGACTCCTA CGAGCAG 27
(2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GCCACCTGGG ACGAGATCTC CTACGAGCAG 30
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GCCACCTGGG ACGGGCAACG TCCACGACCA G 31
(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: GCCACCTGGG TCCGGCCTGG GAGTTACGAG CAG 33
(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GCCACCTGGG ACACCCCATA GACCCCCTGG GGCCAAC 37
(2) INFORMATION FOR SEQ*ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GCCACCTGGG ATGACTCTGG GGCCAAC 27
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRAN EDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GCCCCGCGCG AGTTCCAAGA GACCCAG 27
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GCCACCTGGA CGGGAGGGGG CACAGATACG 30
(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GCCACCTGGG ATGGGCGTAT CGATACTCCC CCTAATAATT CA 42
(2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GCCACCTGGG ACAGGTTCCT AATTCA 26
(2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCCACCTGGG AGAGTTCAAT GAAAAA 26
(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GCCACCTGGG AGAAGGGGGT AGCAATCAGC CC 32
(2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
GCCACCTGGG ACAGGTAGCG GGAACGGTAC GAGCAG 36
(2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: GCCACCTGGG ACAGGCCCCA GCCGGGCGCC GGGGAG 36
(2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GCCACCTGGG AATGCTTCCA GCCCCT 26
(2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GCCACCTGGC ATGTCAAAAA CATT 24
(2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GCCACCGAGT AAGCGGGGGG CCAAGGGAGA TACG 34
(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GCCACCTGGG ATGGGAGTCA GCCC 24
(2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: GCCACCTGGG ACGGCGTCGG GGACCTCGAC GGTAACTAC 39
(2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION; SEQ ID NO: 41: GCCACCTGGG ATGAAGAGGA CCTGAAA 27
(2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GCCACCTGGG ACAGGCCCCT GTCGATGGAT ACG 33
(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GCCACCTGGG ATTCATCCTA CGAGCAG 27
(2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GCCACCTGGG AAAGGGATCT CGGCACAGAT ACG 33
(2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: GCCACCTGGT CCCAGGGGTG GATAATACGA GCAG 34
(2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: GCCACCTGGG ATGTTGAG 18
(2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GCCACCTGGG ATGGATTTTC TAGCACAGAT ACG 33
(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: GCCACCTGGG TGATCTACGA GCAG 24
(2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: GCCACCTGGG ATAGGCGTAA TTCA 24
(2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: GCCACCTGGG ACAGGGTGAG CTCCTACGAG CAG 33
(2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: GCCACCTGGG TACTACAAGA GACCCAG 27
(2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GCCACCTGGG ACCGCCCCAG AAAAA 25
(2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: GCCACCTGGG ACCTCGCCAG GGGCTAC 27
(2) INFORMATION FOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GCCACCCCTA GGAGTAGTGA TTGG 24
(2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: GCCACCTGGG ATGGGAACTC TGGAAACACC 30
(2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: GCCACCTGGG ATGGGCCTTT GGTTGGGGCC AAC 33
(2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: GCCACCTGGG ATGGGCGGAG TGGAAACACC 30
(2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: GCCACCAGTT GGGAGGACCT GAAA 24
(2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: GCCACCTGGG ATGGGAAAGA GACCCAG 27
(2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: GCCACCTGGG ATGGGCGTGG TGATTGG 27
(2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: GCCACCTGGG ATGGCTGAAG CT 22
(2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: GCCACCTTGG AAGAGACCCA G 21
Claims
1. An oligonucleotide that can be used to identify an individual with an increased risk for developing lymphoid leukemia or lymphoma comprising a DNA segment that has a sequence corresponding to that of a region in the genome of said individual wherein said region is within an immune receptor locus wherein said locus is capable of displaying genomic instability due to interlocus rearrangement.
2. The oligonucleotide according to claim 1, wherein said region is within 2000 base pairs of a junction of an inv(7).
3. The oligonucleotide according to claim 2, wherein said oligonucleotide has sufficient homology to hybridize to a VT, Jβ, or Cβ DNA sequence.
4. The oligonucleotide according to claim 3, wherein said oligonucleotide comprises a sequence corresponding to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID N0:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.
5. A method of identifying an individual at an increased risk for developing lymphoid leukemia or lymphoma comprising amplifying a region of immune receptor loci of said individual wherein said loci display genomic instability due to interlocus rearrangement and analyzing products of said amplification for evidence of said genomic instability.
6. The method according to claim 5, wherein said products are analysed by Southern blot analysis.
7. The method according to claim 5, wherein said rearrangement is a TCRτV-TCRβJ rearrangment.
8. The method according to claim 5, wherein said individual has been exposed to a leukemogenic or lymphomagenic agent.
9. The method according to claim 8, wherein said agent is an environmental agent.
10. The method according to claim 8, wherein said agent is a pesticide.
11. The method according to claim 8, wherein said agent is a herbicide.
12. A method of identifying an individual homozygous or heterozygous for the autosomal recessive disease ataxia-telangiectasia or related syndromes comprising amplifying a region of immune receptor loci of said individual wherein said loci display genomic instability due to interlocus rearrangement and analyzing products of said amplificat on for evidence of genomic instability.
13. The method according to claim 12, wherein said products are analyzed by Southern blot analysis.
14. The method according to claim 12 wherein said identification is pre-natal identification.
15. The method according to claim 12, wherein said rearrangement is a TCRτV-TCRβJ rearrangment.
16. A method of identifying a carcinogenic compound comprising exposing peripheral blood or lymphocyte cells to said compound, amplifying a region of immune receptor loci of said cells wherein said loci display genomic instability due to interlocus rearrangement and analyzing products of said amplification for evidence of genomic instability.
17. The method according to claim 16, wherein said products are analyzed by Southern blot analysis.
18. The method according to claim 16, wherein said rearrangement is a TCRτV-TCRβJ rearrangment.
19. 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 the oligonucleotide according to claim 4.
20. A test kit for identifying an individual at an increased risk for developing lymphoid leukemia or lymphoma comprising a container means having disposed therein the oligonucleotide according to claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US68368591A | 1991-04-11 | 1991-04-11 | |
| US683,685 | 1991-04-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1992018648A1 true WO1992018648A1 (en) | 1992-10-29 |
Family
ID=24745044
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1992/002804 WO1992018648A1 (en) | 1991-04-11 | 1992-04-10 | A screening test that identifies individuals at increased risk for the development of lymphoid leukemia and lymphoma |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2014492A (en) |
| WO (1) | WO1992018648A1 (en) |
-
1992
- 1992-04-10 AU AU20144/92A patent/AU2014492A/en not_active Abandoned
- 1992-04-10 WO PCT/US1992/002804 patent/WO1992018648A1/en active Application Filing
Non-Patent Citations (7)
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| AU2014492A (en) | 1992-11-17 |
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