WO2009091879A1 - Réarrangement chromosomique - Google Patents

Réarrangement chromosomique Download PDF

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WO2009091879A1
WO2009091879A1 PCT/US2009/031084 US2009031084W WO2009091879A1 WO 2009091879 A1 WO2009091879 A1 WO 2009091879A1 US 2009031084 W US2009031084 W US 2009031084W WO 2009091879 A1 WO2009091879 A1 WO 2009091879A1
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nucleic acid
probes
array
sample
target nucleic
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PCT/US2009/031084
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Richard Burack
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University Of Rochester
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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
    • 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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Chromosomal abnormalities are a common feature of many diseases and disorders and can occur in both animals and plants. Screening for chromosomal rearrangements is a useful tool in the diagnosis of, for example, many cancers and cancer-predisposing syndromes. Cancer and other genetic-related disorders remain a major cause of morbidity and mortality and there is a continuing need for diagnostic methods that are effective in establishing prognosis and guiding treatment.
  • Described herein are methods, compositions and kits relating to the detection and mapping of chromosomal rearrangements in a biological sample. For example, provided herein is a method for detecting a chromosomal rearrangement in a test nucleotide sequence in a biological sample.
  • the method includes the steps of (a) providing a biological sample including the test nucleotide sequence, wherein the test nucleotide sequence includes a target nucleic acid and a nucleotide sequence 5' to the target nucleic acid; (b)annealing the target nucleic acid to a nucleic acid primer including a nucleotide sequence complementary to the target nucleic acid to form a target-primer hybrid; (c) performing a nucleic acid extension reaction from the target-primer hybrid, wherein the extension reaction produces a labeled nucleic acid sequence complementary to the target nucleic acid and the nucleotide sequence directly 5' of the target nucleic acid; (d) contacting the labeled nucleic acid sequence with a polynucleotide array, wherein the polynucleotide array includes a plurality of addressable probes including at least a first and second set of probes, wherein the first set of probes includes the target nucleic acid, wherein the second set of probe
  • an article of manufacture that includes (a) one or more polynucleotide primers, wherein the polynucleotide primer specifically hybridizes to a target nucleic acid; (b) a polynucleotide array, wherein the polynucleotide array comprises a plurality of addressable probes including at least a first and second set of probes, wherein the first set of probes includes the target nucleic acid and wherein the second set of probes includes the nucleotide sequence 5' of the target nucleic acid; and (c) one or more items selected from the group consisting of packaging material, a package insert with instructions for use, and a container.
  • a database of binding patterns of a plurality of reference samples to a polynucleotide array wherein the polynucleotide array comprises a plurality of addressable polynucleotide probes comprising at least a first and second set of probes, wherein the first set of probes comprises a target nucleic acid, wherein the second set of probes comprise a nucleotide sequence 5' of the target nucleic acid, and wherein a labeled nucleic acid sequence of each reference sample specifically hybridizes to one or more probes in the array.
  • FIG. 1 is a depiction of primer extension from a chromosome 18-specific primer on three different chromosomes: chromosome 14 (top), chromosome 18 (middle) and a chromosome having a translocation between chromosomes 14 and 18 (tl4; 18).
  • FIG. 2 is a depiction of the products obtained from the primer extension reactions in FIG. 1.
  • the heavy lines represent nucleotide segments complementary to chromosome 18; the thin lines represent nucleotide segments complementary to chromosome 14.
  • FIG. 3 is a depiction of array analysis of the products shown in FIG.2 on an oligonucleotide array of chromosome 14-specif ⁇ c probes.
  • the primer extension products that include regions complementary to chromosome 14 bind to the array.
  • FIG. 4 is a graph showing a comparison of Ig kappa usage in five different biological samples.
  • Figure 4A is a normal polyclonal B cell population.
  • Figure 4B is a B cell clone, i.e., a neoplasm, with a rearrangement involving the IgKV 1-8 region.
  • Figure 4C is a B cell clone, i.e., a neoplasm, with a rearrangement involving the IgKV 2-26 region.
  • Figure 4D is a B cell clone, i.e., a neoplasm, with a rearrangement involving the IgKV 1-6 region.
  • Figure 4E is a B cell clone, i.e., a neoplasm, with a rearrangement involving the IgKV 4-1 region.
  • Figure 4F is germline DNA.
  • FIG. 5 is a graph showing a comparison of Ig kappa usage in two individual patients, one in Figure 5 A and the other in Figure 5B.
  • DNA is extracted from biological samples and labeled nucleic acid sequences complementary to Ig kappa regions are obtained by primer extension. The labeled nucleic acids are used to probe a microarray.
  • FIG. 6 is a graph showing a comparison of array detection and localization of translocations involving the IgH and BCL2 loci.
  • Figure 6A shows a novel breakpoint within the BCL2 gene.
  • Figure 6B shows a B cell clone, i.e., a neoplasm, with a breakpoint within the Major Breakpoint Region.
  • Figures 6C and 6D show two different B cell clones, i.e., neoplasms, with different breakpoints within the minor cluster region.
  • Figure 6E shows germline DNA.
  • Chromosomal rearrangements can take a number of different forms. These include deletions, i.e., a portion of the chromosome is missing; translocations, i.e., a portion of one chromosome is transferred to another chromosome; duplications, i.e., a portion of the chromosome is duplicated; amplifications, i.e., a portion of a chromosome is replicated multiple times; and inversions, i.e., a portion of the chromosome is inserted into a chromosome in the opposite orientation of the original portion.
  • the different forms can occur singly or in combination. Regardless of the specific form of the rearrangement, a rearranged chromosome contains a novel DNA sequence created by the juxtaposition and covalent linkage of two DNA segments that are not in physical proximity in the non-rearranged chromosomal homologue.
  • chromosomal rearrangements can disrupt or deregulate the normal expression of genes at the breakpoint, they are often associated with an increased likelihood of the development of particular pathologies. Rearrangements that either disturb the regulation of cellular oncogenes or result in the production of novel proteins with oncogenic properties are a common mechanism of tumorigenesis.
  • the B-cell neoplasm follicular lymphoma
  • IGH immunoglobulin heavy chain
  • the IGH sequences override normal BCL2 gene control elements to drive inappropriately high levels of BCL2 expression, which interfere with normal apoptosis (controlled cell death) of B- lymphocytes.
  • Many other disease-defining rearrangements have been described, including for example, the BCR/ AbI translocation found in chronic myelogenous leukemia (CML); the PML/RAR, the AMLl /ETO and the CBF-beta/MYH translocations found in acute myelogenous leukemia (AML); the EWS/FLI1 translocation found in Ewing's sarcoma; and the MLL/AFF1 and MLL/MLLT3 translocations in acute lymphoblastic and acute myeloid leukemia. Detection of specific rearrangements rapidly directs differential diagnosis for any tumor.
  • the assay involves the synthesis of detectably labeled nucleic acid extension products off the DNA template provided by the biological sample, followed by hybridization of those products to an array.
  • the extension products are synthesized from a primer that has a nucleotide sequence complementary to a specific target nucleotide sequence.
  • the target nucleic acid sequence is one that may be rearranged in the disease or disorder of interest, e.g., the IGH locus in follicular lymphoma.
  • primer extension products include the nucleotide sequence complementary to the target sequence and the nucleotide sequence directly 5' of the target nucleic acid, they can encompass any potentially novel sequence resulting from a chromosomal rearrangement at the location of the target nucleotide sequence.
  • the array is designed to include one or more addressable polynucleotides complementary to the target sequence as well as one or more addressable polynucleotides complementary to the nucleotide sequence 5' of the target sequence.
  • the terms "probe” and “feature” are used interchangeably herein to include selected addressable polynucleotides or combinations of addressable oligonucleotides.
  • the array hybridization pattern indicates the presence or absence of a chromosomal rearrangement.
  • the hybridization pattern also allows for the accurate identification of the specific chromosomal location of the breakpoint.
  • databases of binding patterns are provided.
  • the databases can include the binding patterns of primer extension products synthesized from one or more reference samples.
  • the reference samples can be representative of a wild-type chromosomal arrangement, i.e., the normal or germline chromosomal arrangement, or can be representative of a chromosomal rearrangement.
  • the hybridization pattern generated by primer extension products synthesized from a patient's sample for example, can be compared with standard reference patterns in the database. This information is then used by a skilled artisan, together with other clinical indices, to adjust, if necessary, any treatment regimen the patient may be receiving.
  • the rapid turnaround time afforded by the array hybridization method allows for more efficient identification of specific chromosomal lesions and routine monitoring of an individual patient's response to therapy.
  • the databases can include standard reference patterns from non-human animals or plants. Such databases are useful in the analysis of chromosomal rearrangements in biomedically or agriculturally significant animals and food crops, respectively.
  • a biological sample can be obtained from any prokaryotic or eukaryotic organism.
  • exemplary eukaryotic organisms include, without limitation, mammals, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), pigs, cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice; birds, reptiles, amphibians, or fish, as well as plants, e.g., food crop plants such as corn, rice, wheat or legumes.
  • non-human primates e.g., monkeys, baboons, or chimpanzees
  • pigs cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice
  • birds, reptiles, amphibians, or fish as well as plants, e.g., food crop plants such
  • the biological sample is a clinical sample, e.g., a patient's sample.
  • a "patient's sample” refers to a sample from a human patient and/or a non- human patient.
  • a biological sample can be, without limitation, cells, a cellular sample, a tissue sample, a diagnostic biopsy sample or a fluid sample.
  • cells include, without limitation, peripheral blood mononuclear cells (PBMCs) leukocytes, tissue explants or cell lines.
  • Diagnostic biopsy samples include, for example, lymph node biopsies, tonsil biopsies, bone marrow biopsies, or any biopsy of healthy or diseased tissue.
  • Biological fluid samples include, for example, a blood sample, a plasma sample, a urine sample, a sputum sample, a saliva sample, a lymph sample, or a cerebrospinal fluid sample.
  • Biological samples can be collected from an individual using any standard method known in the art that results in the preservation nucleic acids. Blood samples can be obtained via venous puncture techniques. Serum samples can be prepared from whole blood using standard methods such as centrifuging blood samples that have been allowed to clot. Plasma samples can be obtained by centrifuging blood samples that were treated with an anti-coagulant such as heparin. Saliva samples may be collected using cotton swabs, wipes, suction, or scraping. Biopsies can be collected using standard techniques such as needle biopsy or surgical excision.
  • a biological sample can be assayed for chromosomal rearrangements in a test nucleotide sequence immediately following collection.
  • a biological sample can be stored for later analysis using methods known in the art.
  • a sample can be frozen, dried, freeze-dried or subject to chemical fixation, e.g., formalin fixed and paraffin-embedded.
  • DNA can be extracted from the biological sample using any method in the art that results in DNA that is active in a primer extension reaction.
  • Standard methods of DNA purification include extraction with organic solvents, e.g., phenol, chloroform, isoamyl alcohol or a combination thereof, cesium chloride gradients and others.
  • Commercially available purification reagents and kits can also be used; representative commercial sources include, for example, QIAgen, (Valencia CA) Invitrogen, (Carlsbad, CA) and Stratagene (La Jolla, CA).
  • the purification can be automated. DNA can be partially or substantially purified. In certain cases, "whole genome amplification" is used to increase the amount of DNA available for analysis.
  • Primers sequences will vary according to the gene of interest. Factors governing the choice of specific primers include, but are not limited to, the G/C content of the primer, the primer length desired, the melting temperature of the primer-target DNA, the selectivity of the primer for the target sequence and the location of the corresponding sequences within the DNA of the target nucleic acid. Commercially available primers can be used if the sequences are suitable. Alternatively or in addition, any commercially available primer design program may be used to identify suitable primers for custom synthesis. Of course, the primer length can vary and one of skill in the art will take the factors listed above into account when deciding on the length of any particular primer.
  • nucleic acid extension Any method of nucleic acid extension can be used as long as it generates extension products that are suitable for hybridization to arrays.
  • Primer extension reactions are performed by standard methods (See for example, Ausubel et al., Short Protocols in Molecular Biology, 5 th ed., Wiley & Sons, 2002 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor, NY, 2001.) or using commercially available reagents or kits.
  • Reaction conditions will vary according to a number of factors, including, for example, the primer and target DNA sequence, the length of the extension products desired, the nature of the label, and the specific DNA polymerase that is used.
  • the length of the primer extension product is determined, for example, by the polymerase selected and elongation time for the polymerase; a general model 30 cycles of 60 seconds + 2.5 seconds/ 100 base pairs is a starting point for a skilled artisan.
  • the extension reaction is carried out under linear conditions by varying the amount of time allowed for primer extension or by the addition of chain- terminating nucleotide analogs.
  • the length of the primer extension products will vary according to the location of the primer within the test sequence; all that matters is that the extended products include a sufficient length of the nucleotide sequence directly 5' of the test sequence to effectively hybridize to probes on the array that correspond to the nucleotide sequence directly 5' of the test sequence. Within any given primer extension reaction, the primer extension products will be heterogeneous in length.
  • the skilled artisan may adjust the range of lengths in several ways, including, for example, altering the length of reaction time, shearing the extension products, or by amplifying the products and then partially digesting the amplified extension products with a restriction enzyme that recognizes a cleavage site that occurs relatively frequently in the genome, e.g., a four- base cleavage site, such as an AIu repeat.
  • a restriction enzyme that recognizes a cleavage site that occurs relatively frequently in the genome, e.g., a four- base cleavage site, such as an AIu repeat.
  • the primer extension products include a detectable label.
  • the primer can be labeled directly or indirectly through a covalent linkage at any one, two, three or four or more nucleotides.
  • the primer extension products can be labeled using any method known in the art; the choice of a particular method will vary according to many factors, including, for example, the length and nucleotide sequence of the extension product, the efficiency of the extension reaction, the structure and size of the label, the efficiency of incorporation of the label into the extension product and the detection systems used.
  • one or more labeled precursors can be added to the primer extension reaction and incorporated into the primer extension products.
  • the label can be post-synthetically added to the primer extension products.
  • a modified dNTP e.g., amino-allyl modified dUTP, can be used for synthesis of the primer extension product and a label attached post-synthetically via the free amine.
  • Useful detectable labels include but are not limited to, a fluorophore, e.g., xanthene dyes, e.g. fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6- carboxy-2',4',7',4,7-hexachloro fluorescein (HEX), 6-carboxy-4', 5'-dichloro-2', T- dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G.sup.5 or G5), 6-carboxyrhodamine-6G (R6G.sup.6 or G.sup.6)
  • umbelliferone benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g., Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., eyanine dyes such as Cy3, Cy5, etc; BODIPY dyes, ALEXA dyes and quinoline dyes; a biotin moiety, which is subsequently bound by a labeled avidin or streptavidin molecule, a chemiluminescent label, a bioluminescent label, a hapten, a metal chelator or an enzyme.
  • benzimide dyes e.g. Hoechst 33258
  • phenanthridine dyes e.g., Texas Red
  • ethidium dyes e.g., Texas Red
  • the primer extension products can be subjected to one or more cycles of amplification.
  • the primer extension products may be partially or substantially purified prior to the amplification step.
  • the purification step can be carried out by standard methods known in the art. (See, for example, Ausubel et al., Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor, NY, 2001.) Any amplification method known in the art that results in nucleic acids suitable for hybridization to an array can be used.
  • primer extension products are annealed with one or more primer sequences and amplified by polymerase chain reaction (PCR) in the presence of a detectable label as described above.
  • PCR polymerase chain reaction
  • Useful DNA polymerases include, for example, Taq DNA polymerase, modified Taq DNA polymerases or other DNA polymerases in which the 3' exonuclease activity has been attenuated or eliminated relative to that of the wild type polymerase, e.g., exo-Pfu DNA polymerase or exo-Klenow fragment.
  • a detectable label can be incorporated into the amplification product, either during amplification or post- synthetically using the methods and labels described above.
  • the amplification product can be labeled by including modified nucleotides in the amplification reaction, e.g., amino-allyl modified dUTP, which are then labeled post- synthetically through the free amine.
  • the second primer sequences can be complementary to a known sequence within the primer extension product or can be a random sequence, e.g., random hexamers, random heptamers, random octamers or the like.
  • the primers can include a detectable label or a modified nucleotide that can be used for post-synthetic labeling of the amplification product.
  • a specific primer sequence can be ligated to one end of the primer extension product using, for example, T4 RNA ligase, and the primer extension product amplified by ligation-mediated PCR,
  • array platforms can be used as long as probes comprising the target nucleic acid and the nucleic acid directly 5' of the target nucleic acid, i.e., probes complementary to the primer extension product are sufficiently represented in the array.
  • Array platforms are generally well known in the art ⁇ e.g., see Pinkel et al., Nat. Genet. (1998) 20:207-211 ; Hodgson et al., Nat. Genet. (2001) 29:459-464; Wilhelm et al., Cancer Res. (2002) 62: 957-960).
  • the array can be fabricated using any method known in the art.
  • An array can be a microarray (also commonly known as gene or genome chip, DNA chip, or gene array), i.e., a collection of microscopic DNA spots, generally representing single polynucleotide sequences, arrayed on a solid surface by covalent attachment to a chemical matrix.
  • the density of the probes in the array will vary depending upon the sequence of interest. Typical high density arrays include a probe every 1000 base pairs in the human genome or as many as 2.1 x 10 6 probes per array.
  • useful arrays contain a plurality ⁇ i.e., at least about 100, at least about 500, at least about 1000, at least about 2000, at least about 5000, at least about 10,000, at least about 20,000, usually up to about 100,000 or more) of addressable probes containing polynucleotides that are linked to a usually planar solid support via one terminus of the polynucleotide molecules ⁇ i.e. either the 3' or 5' end).
  • Low density arrays can have the same probe-spacing density as high density arrays, but do not attempt to sample the entire genome. For example, they can be designed to thoroughly interrogate regions of interest, e.g., BcI genes, the IGH@, IGK@, IGL@.
  • Such arrays are typically include less than about 500 gene regions for highly specific interrogation of genes of interest.
  • the polynucleotide molecules may vary in length and origin and can include, for example, oligonucleotides, PCR products, bacterial artificial chromosomes(BACs), cDNAs, or inserts from phage or plasmids.
  • the polynucleotides can be about 10-25 bases in length, 15-80 bases in length, about 20-100 bases in length, about 100-1000 bases in length, or about 200-5000 bases in length, depending on the platform used.
  • useful oligonucleotide probes for high density arrays that are fine-tiled through the region of interest can be about 70 bases; useful PCR probes for low-density arrays can be about 500-700 bases in length. Suitable array platforms and exemplary methods by which such assays may be performed, are described in issued U.S. Pat. Nos.
  • the array can be designed so that all the probes have matching annealing requirements, i.e., all the probes effectively anneal with their respective target nucleotide sequences under substantially similar conditions, for example, similar temperatures, and in solutions of similar ionic concentration.
  • the arrays can be commercially available arrays that include standard probe sets; alternatively or in addition, arrays can be custom designed so that the probe sets are more precisely tailored to the test sequence of interest.
  • arrayIT Single-stranded DNA
  • Agilent Technologies Santa Clara, CA
  • Affymetrix Santa Clara, CA
  • GE Healthcare Primary Healthcare
  • Standard hybridization techniques are used to hybridize a sample to an array. Suitable methods are described in (Kallioniemi et al., Science 258:818-821 (1992) and WO 93/18186). Several guides to general techniques are available, e.g., Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993).
  • the hybridization methods encompass the steps of (1) immobilization of polynucleotides on a solid support by one end of the oligonucleotide; or spotting of DMSO-denatured PCR products, followed by UV crossing-linking; (2) pre-hybridization treatment to increase accessibility of support-bound polynucleotides and to reduce nonspecific binding; (3) hybridization of a mixture of labeled primer extension products to the surface-bound nucleic acids, typically under high stringency conditions; (4) post- hybridization washes to remove nucleic acid fragments not tightly bound to the solid support polynucleotides; and (5) detection of the hybridized labeled nucleic acids.
  • the reagents used in each of these steps and their conditions for use vary depending on the particular application. More specifically, the stringency of the hybridization conditions may vary and are adjusted by the skilled artisan to conditions that limit the binding of the primer extension products to specifically complementary probes. High stringency assay conditions are described in, for example, Ausubel et al., Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor, NY, 2001.
  • Hybridization patterns are detected using standard methods and equipment.
  • a fluorescence reader is used to detect the hybridization pattern represented by the fluorescent labels. Analysis is performed by comparing the pattern to a database of known patterns.
  • a reference sample is used to determine whether a particular hybridization signal intensity is present, reduced, normal, or elevated relative to a control sample or a larger population.
  • a reference chart can contain the normal range of a particular level of hybridization signal intensity found in healthy individual of the same age, ethnic background or general health as the individual in question. Using this reference chart, any level of hybridization signal intensity measured in a sample can be classified as being present, reduced, normal, or elevated relative to a control sample or a larger population.
  • the level of a hybridization signal intensity in a biological sample can be "normalized” against the level of one or more additional biological markers, for example another genetic locus, whose expression is known to be independent of the chromosomal rearrangement of interest. That is, the levels of the additional marker can be evaluated in parallel with those of the hybridization signal intensity of interest, either at the same time or on a separate occasion.
  • the additional marker can serve as an internal control for sample preparation, handling and storage as well as day-to-day assay variability.
  • the values for the level of the hybridization signal intensity of interest and the additional marker may be expressed as a ratio and the ratios may be compared to similar ratio obtained for a reference sample or population.
  • the individual's relative risk for a chromosomal rearrangement can be assessed. Any statistical method known in the art for evaluating relative risk may be used.
  • a computer system comprising a database of hybridization patterns characteristic of a specific chromosomal rearrangement or the absence of a specific chromosomal rearrangement pattern, for example, the BCR/ AbI translocation found in chronic myelogenous leukemia (CML); the PML/RAR, the AMLl /ETO and the CBF-beta/MYH translocations found in acute myelogenous leukemia (AML); the EWS/FLI1 translocation found in Ewing's sarcoma; the MLL/AFF1 and MLL/MLLT3 translocations in acute lymphoblastic and acute myeloid leukemia; translocations found in lymphomas, e.g., non-Hodgkin lymphoma, including for example, the IGH/Bcl2 translocation found in follicular lymphoma, the IGH/Bcl6 translocation found in diffuse large B-cell lymphoma, the IGH/myc translocation found
  • CML chronic
  • translocations that can be included in the database are described in Bende et ah, (2007) Leukemia, Jan;21(1): 18-29 and in the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue, 4th edition, edited by Swerdlow et al., (2008) Published by the International Agency for Research on Cancer, (IARC) 69008 Lyon, France, which are herein incorporated by reference in their entireties, at least for translocations taught therein.
  • the computer system also included a user interface capable of determining matches between a sample and the database for displaying the results.
  • Also provided is a machine-readable medium including program instructions for performing the following steps: 1 ) comparing the hybridization pattern of a test sample obtained using the methods taught herein with a control hybridization pattern that correlates with the presence or absence of a chromosomal rearrangement, and 2) providing an outcome as to the presence or absence of a chromosomal rearrangement in the test sample.
  • Assay Applications 1 ) comparing the hybridization pattern of a test sample obtained using the methods taught herein with a control hybridization pattern that correlates with the presence or absence of a chromosomal rearrangement, and 2) providing an outcome as to the presence or absence of a chromosomal rearrangement in the test sample.
  • disorders associated with chromosomal rearrangements include hematologic disorders and cancers such as acute myelogenous leukemias, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, Ewing's sarcoma, Hodgkin lymphoma, non-Hodgkin lymphomas (such as diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, marginal zone lymphoma), myeloma and other plasma cell malignancies, malignant melanoma, desmoplastic small round cell tumor, liposarcoma, papillary thyroid carcinoma, alveolar rhabdomyosarcoma, angiomatoid malignant fibrous histiocytoma (AMFH),
  • hematologic disorders and cancers such as acute myelogenous leukemias, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute lymphoblast
  • nucleic acid suspected of having been involved in a chromosomal rearrangement can be analyzed using the methods herein.
  • the nucleic acid i.e., the test sequence
  • test sequences for cancer and hematologic cell disorders include, but are not limited to, ABL, ALK, AMLl , API, BCL2, BCL3, BCL6, BCLlO, BCR, CARS, CBF- ⁇ , CCNDl, CLTC, CRTCl (METC), CDK4, CDKN1A/B, CDKN2A/B, DDIT, ERG, ETO, ETVj , ETV 4 EWS, FAS(CD95), FEV, FKHR, FLIl , FUS, HMGA2, IGH@, IGK@, IGL@, MALTl, MAML2, MLL, MYC, MYHI l, NPM, NRTKl, PAX3, PAX5, PAX7, PBXl, PLAGl, PML, RANBPl, RARA, RET, RPS17, SSXi, SSX 2 , SYT, TCF3, TEL, TPM3, TPM4, TP53, TRA@ TRB
  • the test sequence can include one or more nucleotide sequences that are known to be rearranged in FL, e.g., IGH@, IGK@, IGL@, BCLl, BCL2, BCL3, BCL6, BCLlO, CCNDl , MALTl , or PAX5.
  • Useful test sequences for FL include IGH@ and BCL2.
  • a representative sequence for IGH@ (also referred to as, for example, IGH; IGH.1@; IGHDYl ; cold agglutinin FS-I H-chain - fragment; cold agglutinin FS-2 H-chain - fragment; IGH locus; immunglobulin heavy chain precursor - fragment; immunoglobulin heavy locus; immunoglobulin heavy polypeptide gene cluster (V,D,J,C)) includes NG_001019.
  • test sequences for BCL2 include NM 000633 [gi:72198188] and NP_000648 [gi: 72198346].
  • the test sequence can include one or more nucleotide sequences that are known to be rearranged in CML, e.g., BCR and ABL.
  • Representative sequences for BCR include NMJ)04327[gi:82546842] and NM_021574 [gi: 82546844].
  • a representative sequence for ABL also referred to as EC 2.7.10.2, JTK7, c-ABL, pi 50, Abelson murine leukemia viral (v-abl) oncogene homolog, and proto-oncogene tyrosine-protein kinase ABLl
  • the test sequence can include one or more nucleotide sequences that are known to be rearranged in acute leukemias, e.g., BCR, ABLl, MLL, TEL(ETVo), AMLl (RUNXl), E2A (TCF3), PBX1,IGH@, IL3, PML, RAR, AMLl, ETO, CBF-beta and MYH.
  • the test sequence can include one or more nucleotide sequences that are known to be rearranged in B-cells, e.g., IGK@.
  • IGK@ include NG_000833 [gi:19743567] and NG_OOO83 [gi: 19718803].
  • the test sequence can include one or more nucleotide sequences that are known to be rearranged in Ewing's sarcoma, e.g. EWS, FLIl, ERG, ETVl, ETV4, and FEV.
  • EWS include NM 005243 [gi: 125987606] and NM_013986 [gi:125987607].
  • a test sequence can also be an exogenous sequence that is known to integrate within the genomic DNA of a host.
  • the exogenous sequence can be a viral sequence, for example, Epstein-Barr virus (EBV), HHV-8, SV-40 or adenovirus.
  • EBV Epstein-Barr virus
  • HHV-8 HHV-8
  • SV-40 adenovirus
  • adenovirus a viral sequence that may exert their pathologic effects by disruption of specific cellular genes in the host.
  • EBV associated with Hodgkin lymphoma and nasopharyngeal carcinoma.
  • the test sequence can include one or more EBV nucleotide sequences.
  • HHV-8 is associated with plasma cell malignancies and Castleman disease.
  • the test sequence can include one or more HHV-8 nucleotide sequences.
  • the exogenous sequence can also be a transposable element, i.e., a mobile genetic element that contributes to genetic variability in plants by randomly integrating within the plant genome.
  • a labeled primer extension product complementary to a test nucleotide sequence and the nucleotide sequence directly 5' of the test nucleotide sequence in the biological sample is synthesized and hybridized to an array.
  • the particular hybridization pattern is indicative of the absence or presence of a chromosomal rearrangement.
  • the primer extension products will anneal to probes corresponding to the test sequence and to those corresponding to sequences directly 5' of the test sequence.
  • the primer extension products will anneal to probes corresponding to the test sequence and not to the probes corresponding to sequences directly 5' of the test sequence in its native location.
  • a test sequence that has undergone a chromosomal rearrangement generates a novel hybridization signal at a probe or set of probes corresponding to the nucleic acid sequence located directly 5' of the rearranged test sequence. If the chromosomal location of those probes is known, the precise chromosomal location of the rearrangement can be determined. By including a series of overlapping probes, e.g. oligonucleotides, the fine structure of the breakpoint can be precisely mapped.
  • primer extension and array analysis method is also amenable to use in a multiplex format using primer "cocktails" which include primers that are complementary to two or more test sequences that are known to be rearranged in specific diseases or disorders.
  • primer "cocktails” which include primers that are complementary to two or more test sequences that are known to be rearranged in specific diseases or disorders.
  • the materials and methods provided herein are useful for comparing the patterns of chromosomal rearrangement in multiple samples, for example, clinical samples from a patient population.
  • the methods are also useful for comparing the patterns in a single individual over time, for example, for monitoring the tumor burden over time, assisting the skilled artisan in the management of certain tumors , e.g., follicular lymphoma, for which a "watchful waiting" strategy may be advised or for monitoring the response of a patient during a course of therapy, or for monitoring a patient who has or is suspected of having an autoimmune, inflammatory, or infectious disorder.
  • the materials and methods provided herein can also be used to monitor chromosomal rearrangements in domestic or laboratory animals, e.g., during preclinical drug discovery and development.
  • Laboratory animals can be any species of animal used by those of ordinary skill in the art for the study of such disorders and can include, without limitation, non-human primates, dogs, cats, rats and mice.
  • the materials and methods provided herein can also be used to monitor chromosomal rearrangements in plants, particularly in agriculturally significant crop plants.
  • kits comprising a database of binding patterns as described herein.
  • Optional or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • Example 1 Detection of deletions within the Ig Kappa locus: Comparison of B-cell tumors with normal samples
  • the Ig kappa variable gene locus includes 76 genes; during B cell development, a deletion occurs between the J-gene and one of these kappa variable genes, resulting in the juxtaposition of these two genetic regions.
  • Deletions within the human Ig kappa locus in patients with B cell neoplasia are analyzed by probing microarrays with primer extension products complementary to the Ig kappa locus.
  • the microarray data are compared with deletion analysis performed by include cloning and sequencing of the rearranged IGKV region using standard currently available PCR methods.
  • Biological samples are collected from four individual patients who have been diagnosed with a B cell malignancy.
  • the biological samples are diagnostic biopsies, aspirates or peripheral blood mononuclear cells (PBMCs). Normal control samples are collected from tonsils and PBMCs.
  • DNA extractions and primer extension reactions are performed using standard techniques. (See, for example, Ausubel et al., Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor, NY, 2001.)
  • the primer extension reactions are carried out under linear conditions.
  • the primers are Jh-region consensus primers for the IGK locus.
  • Primer extension products are analyzed on a microarray having 4576 unique features, each about 60 base pairs long, derived from the plus strand of the 474,006 base pair region of human chromosome 2 that spans the J gene and V gene regions of the Ig kappa light chain locus.
  • extension products from a normal polyclonal B cell population, which includes numerous related clones, each having a unique deletion generate multiple signals, as shown in Figure 4A.
  • the extension products from specific B-cell neoplasias generate unique patterns based on the location of the deletion, as shown in Figures 4B, 4C, 4D, and 4E.
  • Figure 4B is a B cell clone with a rearrangement involving the IgKV 1-8 region.
  • Figure 4C shows a B-cell clone with a rearrangement involving the IgKV 2-26 region.
  • Figure 4D is a B cell clone with a rearrangement involving the IgKV 1-6 region.
  • Figure 4E is a B cell clone with a rearrangement involving the IgKV 4-1 region.
  • B-cell repetoires Biological samples are collected from two individual patients who have been diagnosed with a B cell malignancy. DNA extractions are performed using standard techniques. In order to determine the relative abundance of B cells with each deletion, the primer extension reactions are carried out under linear conditions conditions by varying the amount of time allowed for primer extension or by the addition of chain-terminating nucleotide analogs. The primers are Jh-region consensus primers for the IGK locus. The primer extension products are analyzed on a microarray as described in Example 2.
  • follicular lymphoma In follicular lymphoma, the location of the translocation between the IGH locus and BCL2 genes (on chromosomes 14 and 18, respectively) has prognostic value. Detection and localization of the t(l 4: 18) translocation in patients with follicular lymphoma is performed by probing microarrays with primer extension products extending from the J genes in the IGH locus. The microarray data are compared with an analysis performed by cloning and sequencing of the translocated IGH/BCL2 locus using standard, currently available PCR methods. Biological samples are collected from six individual patients who have been diagnosed with a follicular lymphoma. Normal non-lymphoid control samples are collected from three individuals.
  • DNA extractions and primer extension reactions are performed as described above.
  • the primer extension reactions are carried out under linear conditions.
  • the primers are Jh-region consensus primers for the IGH locus.
  • the primer extension products are hybridized to a microarray having 581 unique features, each about 60 base pairs long, derived from the plus strand of the 400028 base pair region of human chromosome 18 that spans the BCL2 locus.
  • Figue 6A is a B- cell clone with a novel translocation involving the IGH locus and a region of the BCL2 locus that is outside of the regions typically associated with breakpoints.
  • Figure 6B is a B-cell clone with a translocation involving the IGH locus and the MBR of BCL2.
  • Figure 6C is a B-cell clone with a translocation involving the IGH locus and the mcr, i.e., the mbr, of BCL2;
  • Figure 6D is a B-cell clone with a translocation involving the IGH locus and the mcr, i.e., the mbr, of BCL2.
  • Comparison of Figures 6C and 6D indicates that, although both B-cell clones contain translocations within the mcr, the precise locations of the breakpoints in the chromosomes differs.

Abstract

L'invention concerne des matériaux et des procédés pour la détection et la cartographie de réarrangements chromosomiques dans un échantillon biologique. L'analyse implique de manière générale la synthèse de produits d'extension d'acides nucléiques marqués de manière détectable en dehors de la matrice d'ADN fournie par l'échantillon biologique suivi de l'hybridation de ces produits vers une puce.
PCT/US2009/031084 2008-01-18 2009-01-15 Réarrangement chromosomique WO2009091879A1 (fr)

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CN104894111A (zh) * 2015-05-18 2015-09-09 李卫东 用于白血病染色体畸变高通量测序的dna靶向捕获阵列
CN106191262A (zh) * 2016-07-18 2016-12-07 南开大学 一种分析Igκ基因二次重排中间产物的方法及试剂盒
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011050981A3 (fr) * 2009-10-30 2011-12-08 Roche Diagnostics Gmbh Procédé de détection d'aberrations chromosomiques équilibrées dans un génome
CN104894111A (zh) * 2015-05-18 2015-09-09 李卫东 用于白血病染色体畸变高通量测序的dna靶向捕获阵列
CN106556581A (zh) * 2015-09-24 2017-04-05 无锡源清天木生物科技有限公司 等离子体对体外dna断裂损伤强度的快速检测方法
CN106556581B (zh) * 2015-09-24 2020-10-27 无锡源清天木生物科技有限公司 等离子体对体外dna断裂损伤强度的快速检测方法
CN106191262A (zh) * 2016-07-18 2016-12-07 南开大学 一种分析Igκ基因二次重排中间产物的方法及试剂盒
CN113811623A (zh) * 2019-05-16 2021-12-17 北京市肿瘤防治研究所 定量检测人cdkn2a基因拷贝缺失的方法、引物及其用途
CN114657254A (zh) * 2022-05-17 2022-06-24 至本医疗科技（上海）有限公司 用于bcr/tcr基因重排检测的试剂盒和装置

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