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WO1998005677A1 - Mutation du gene de susceptibilite du cancer du sein et de l'ovaire - Google Patents

Mutation du gene de susceptibilite du cancer du sein et de l'ovaire Download PDF

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Publication number
WO1998005677A1
WO1998005677A1 PCT/US1997/013654 US9713654W WO9805677A1 WO 1998005677 A1 WO1998005677 A1 WO 1998005677A1 US 9713654 W US9713654 W US 9713654W WO 9805677 A1 WO9805677 A1 WO 9805677A1
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Prior art keywords
nucleotide sequence
seq
oligonucleotide
allele
mutation
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PCT/US1997/013654
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English (en)
Inventor
Patricia D. Murphy
Antonette C. Allen
Marga B. White
Sheri J. Olson
Bin Zeng
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Oncormed, Inc.
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Publication date
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Priority to AU40509/97A priority Critical patent/AU4050997A/en
Publication of WO1998005677A1 publication Critical patent/WO1998005677A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • This invention relates to gene mutations that predispose individuals to breast and ovarian cancer. More specifically, this invention relates to specific mutations in the BRCA1 gene. In addition, it also relates to methods for detecting the presence of these mutations.
  • genomic DNA is extracted from whole blood and the coding regions of the BRCA1 gene are amplified using the polymerase chain reaction (PCR). Each of the coding regions is sequenced completely and the results are compared to the normal DNA sequence of the gene (GenBank Accession Number U14680). Many mutations have already been reported in the BRCA1 gene. Shattuck- Eidens, D., et al, Journal of the American Medical Association 273: 535-541 (1995).
  • the BRCA1 gene (GenBank Accession Number U14680) is divided into 24 separate exons. Exons 1 and 4 are noncoding, in that they are not part of the final functional BRCA1 protein product.
  • the BRCA1 coding region spans roughly 5600 base pairs (bp). Each exon consists of 200-400 bp, except for exon 11 which contains about 3600 bp.
  • To sequence the coding region of the BRCA1 gene each exon is amplified separately and the resulting PCR products are sequenced in the forward and reverse directions. Because exon 11 is so large, we have divided it into twelve overlapping PCR fragments of roughly 350 bp each (segments "A" through "L'Of BRCAl exon ll).
  • the present invention is based on the discovery of specific mutations of the published BRCAl cDNA sequence which are associated with susceptibility to and development of breast and ovarian cancer. It is an object of the invention to provide a method for determining a predisposition or higher susceptibility to breast and ovarian cancer.
  • the invention provides, an allele-specific oligonucleotide (especially, a labeled oligonucleotide) for detecting a mutation in a BRCAl encoding target polynucleotide, wherein the allele-specific oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the detection of a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.
  • the invention also provides a primer oligonucleotide for amplifying a region of a BRCAl-encoding target polynucleotide, wherein the primer oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the amplification of a portion of the target polynucleotide that includes nucleotides that flank nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159, or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.
  • the invention is particularly directed to the embodiment wherein the detected mutation is a 2799delAA, a 4158delAG mutation, a 5053delG mutation and /or a 943insl0 mutation.
  • the invention additionally provides a method for classifying a tumor for diagnostic and prognostic purposes or detecting a predisposition of higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating a BRCAl encoding target polynucleotide from an individual; and b) determining the identity of at least one nucleotide in a polymorphic region selected from the group of nucleotides corresponding to nucleotides at positions 943, 944, 2799, 2800, 4158, 4159, and 5053 of a BRCAl cDNA sequence having the nucleotide sequence of Genbank Accession Number U14680; and c) classifying the BRCAl-encoding target polynucleotide for the diagnostic and prognostic purposes
  • BRCAl-encoding target polynucleotide for diagnostic and prognostic purposes of detecting a predisposition or higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating the BRCAl-encoding target polynucleotide from an individual; b) incubating the target polynucleotide in the presence of an allele- specific oligonucleotide, wherein the incubation is under conditions sufficient to allow hybridization to occur between the target polynucleotide and the allele-specific oligonucleotide; wherein the allele-specific oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the detection of a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank
  • the invention is particularly directed to the embodiment of such methods wherein the detected mutation is a 2799delAA, a 4158delAG mutation, a 5053delG mutation and/or a 943insl0 mutation.
  • the invention additionally provides a kit, comprising a carrier means being compartmentalized to receive in close confinement one or more container means, the container means comprising an allele-specific oligonucleotide; wherein the allele-specific oligonucleotide has a nucleotide sequence sufficient to permit the oligonucleotide to specifically hybridize to a BRCAl-encoding polynucleotide, and to detect a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.
  • the BRCAl gene is a tumor suppressor gene associated with breast and ovarian cancer.
  • the present invention is based on the discovery of the significance of mutations in the BRCAl cDNA sequence, and in particular, of the significance of certain specific mutations in the BRCAl cDNA sequence. These mutations are referred to as 2799delAA, 4158delAG, 5053delG, and 943insl0, respectively.
  • the 2799delAA mutation is a two base pair deletion of nucleotides
  • the 4158delAG mutation is a two base pair deletion of nucleotides
  • the 5053delG mutation is a one base pair deletion of nucleotide
  • the 943insl0 mutation is a 10 base pair tandem repeat insertion between nucleotides 943 and 944, which interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 289. This mutation is also predicted to result in a truncated, and most likely, non-functional protein.
  • Useful polynucleotides (especially DNA molecules) according to the invention are those which will specifically hybridize to BRCAl sequences in the region of the 2799delAA, the 4158delAG, the 5053delG, or the
  • 943insl0 mutation such that, by virtue of such hybridization, or by polymerase-mediated extension, or by ligation with other polynucleotides, permit the identification of the nucleotides present (or deleted from) the region of the 2799delAA, the 4158delAG, the 5053delG, or the 943insl0 mutation.
  • polynucleotides will be DNA molecules that are at least about 20 nucleotides in length and have a nucleotide sequence corresponding to a region of the published BRCAl cDNA sequence
  • DNA molecules contain base changes corresponding to the 2799delAA, 4158delAG, 5053delG, or 943insl0 mutations.
  • Such molecules can be used as allele-specific oligonucleotide probes to track a particular mutation through a family.
  • Body samples can be tested to determine whether the BRCAl gene contains one or more of the mutations 2799delAA, 4158delAG, 5053delG, or 943insl0.
  • Suitable body samples for testing include those comprising
  • DNA, RNA or protein obtained from biopsies, blood, prenatal, or embryonic tissues, for example.
  • pairs of isolated oligonucleotide primers are provided.
  • suitable pairs of oligonucleotide primers include:
  • BRCA1-11G-F 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l), and
  • BRCA1-11G-R 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2).
  • BRCA1-11L-F 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3), and
  • BRCA1-11L-R 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ ID NO:4).
  • BRCA1-16-F 5'- AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5), and
  • BRCA1-16-R 5'-AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6).
  • BRCA1-11A-F 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7)
  • BRCA1-11A-R 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8).
  • BRCA1-11G refers to a sequence in the BRCAl gene, Exon 11, section G
  • BRCA1-11L refers to a sequence in the BRCAl gene, Exon 11, section L
  • BRCA1-16 refers to a sequence in the BRCAl gene, Exon 16
  • BRCA1-11L refers to a sequence in the BRCAl gene, Exon 11, section A.
  • F and R refer to forward and reverse.
  • the oligonucleotide primers are useful in diagnosis of a subject at risk of having breast or ovarian cancer, and are also useful for characterizing a tumor.
  • the primers direct amplification of a target polynucleotide prior to sequencing.
  • These unique BRCAl oligonucleotide primers were designed and produced at OncorMed based upon identification of the 2799delAA, 4158delAG, 5053delG, and 943insl0 mutations.
  • isolated allele specific oligonucleotides are provided.
  • the allele specific oligonucleotides are useful in diagnosis of a subject at risk of having breast or ovarian cancer, and also useful for characterizing a tumor.
  • the term “substantially complementary to” or “substantially the sequence” refers to (e.g., SEQ ID NO:9 and SEQ ID NO:10) sequences which hybridize to the sequences provided under stringent conditions and /or sequences having sufficient homology with SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:ll and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, or SEQ ID NO:15 and SEQ ID NO:16 such that the allele specific oligonucleotides of the invention hybridize to the sequence, under conditions in which the non-allele oligonucleotides are substantially less capable or incapable of hybridizing.
  • isolated includes oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they may be associated. Such association being either in cellular material or in a synthesis medium.
  • a "target polynucleotide” refers to a nucleic acid sequence of interest e.g., the BRCAl encoding polynucleotide.
  • Other primers which can be used for primer hybridization will be known or readily ascertainable to those of skill in the art.
  • primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a significant number of nucleic acids in the polymorphic locus.
  • primer refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight and most preferably at least about 20 nucleotides of the BRCAl gene wherein said DNA sequence contains the 2799delAA mutation relative to BRCAl contained in SEQ ID NO's: 9 and 10; the 4158delAG mutation relative to
  • Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be of sufficient length to prime the synthesis of extension products in the presence of the inducing agent for polymerization.
  • primers of the invention are designed to be "substantially" complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the mutation to hybridize therewith and permit amplification of the genomic locus.
  • Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved.
  • one primer is complementary to the "negative" (-) strand of the polymorphic locus and the other is complementary to the "positive” (+) strand.
  • Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA polymerase I (Klenow) and nucleotides results in newly synthesized + and - strands containing the target polymorphic locus sequence.
  • the product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
  • the oligonucleotide primers of the invention may be preferably prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.
  • diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., Tetrahedron Letters, 22:1859-1862, 1981.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
  • Any nucleic acid specimen, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, providing it contains, or is suspected of containing, the specific nucleic acid sequence containing the polymorphic locus.
  • the process may amplify, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded.
  • DNA or RNA may be single stranded or double stranded.
  • enzymes, and /or conditions optimal for reverse transcribing the template to DNA would be utilized.
  • a DNA-RNA hybrid which contains one strand of each may be utilized.
  • a mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so utilized.
  • the specific nucleic acid sequence to be amplified i.e., the polymorphic locus, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.
  • DNA utilized herein may be extracted from a body sample, such as blood, tissue material and the like by a variety of techniques such as that described by Maniatis, et. al. In: Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, NY, p 280-281, 1982). If the extracted sample is impure, it may be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
  • the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to denature the double-stranded molecules (e.g., about 90°C-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes). After this heating period, the solution is allowed to cool to conditions that are preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable.
  • This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
  • the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
  • the agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E.
  • coli DNA polymerase e.g., coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase.
  • heat-stable enzymes i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation
  • Taq polymerase e.g., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation
  • Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand.
  • the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
  • the newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described above and this hybrid is used in subsequent steps of the process.
  • the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
  • the amplification products may be detected by analyzing it by Southern blots without using radioactive probes.
  • a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a
  • probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
  • a radioisotope for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
  • Sequences amplified by the methods of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et. al, Proc. Natl. Acad. Sci. (U.S.A.)., 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landgren, et. al, Science, 241:1007, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landgren, et. al, Science, 242:229-237, 1988).
  • the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art.
  • Alternative methods of amplification have been described and can also be employed as long as the BRCAl locus amplified by PCR using primers of the invention is similarly amplified by the alternative means.
  • Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter. Reverse transcriptase copies the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA.
  • nucleic acid sequence-based amplification is nucleic acid sequence-based amplification (NASBA) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme.
  • NASBA can begin with either DNA or RNA and finish with either, and amplifies to 10 8 copies within 60 to 90 minutes.
  • nucleic acid can be amplified by ligation activated transcription (LAT).
  • LAT ligation activated transcription
  • Amplification is initiated by ligating a cDNA to the promoter oligonucleotide and within a few hours, amplification is 10 8 to 10 9 fold.
  • the QB replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag- along sequence of interest.
  • Another nucleic acid amplification technique ligase chain reaction (LCR), works by using two differently labeled halves of a sequence of interest which are covalently bonded by ligase in the presence of the contiguous sequence in a sample, forming a new target.
  • the repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences.
  • a 2-base gap separates the oligonucleotide probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair.
  • Nucleic acid amplification by strand displacement activation (SDA) utilizes a short primer containing a recognition site for Hindi with short overhang on the 5' end which binds to target DNA.
  • SDA strand displacement activation
  • DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs. Hindi is added but only cuts the unmodified DNA strand. A DNA polymerase that lacks 5' exonuclease activity enters at the cite of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer. SDA produces greater than 10 7 -fold amplification in 2 hours at 37°C. Unlike PCR and LCR, SDA does not require instrumented temperature cycling. Another amplification system useful in the method of the invention is the QB Replicase System. Although PCR is the preferred method of amplification if the invention, these other methods can also be - lo ⁇
  • a method for diagnosing a subject having a predisposition or higher susceptibility to (at risk of) breast or ovarian cancer comprising sequencing a target nucleic acid of a sample from a subject, by the dideoxynucleotide sequencing ("dideoxy sequencing") method (Sanger, F., et al, J. Mol Biol. 142:1617 (1980), herein incorporated by reference), following amplification of the target nucleic acid.
  • dideoxy sequencing dideoxynucleotide sequencing
  • a method for diagnosing a subject having a predisposition or higher susceptibility to (at risk of) breast or ovarian cancer comprising contacting a target nucleic acid of a sample from a subject with a reagent that detects the presence of one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 and detecting the mutation.
  • a method for characterizing a tumor comprises sequencing the target nucleic acid isolated from the tumor to determine if one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 has occurred. Another method comprises contacting a target nucleic acid of a sample from a subject with one or more reagents that detect the presence of one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 and detecting the mutation.
  • a number of hybridization methods are well known to those skilled in the art. Many of them are useful in carrying out the invention.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • container means such as vials, tubes, and the like
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise means for amplifying BRCAl DNA, said means comprising the necessary enzyme(s) and oligonucleotide primers for amplifying said target DNA from the subject.
  • the oligonucleotide primers include primers having a sequence:
  • BRCA1-11G-F 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l)
  • BRCA1-11G-R 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2)
  • BRCA1-11L-F 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3)
  • BRCAl-llL-R 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ ID NO:4), or
  • BRCA1-16-F 5'-AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5)
  • BRCA1-16-R 5'- AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6) or
  • BRCA1-11A-F 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7)
  • BRCA1-11A-R 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8)
  • the targets flanking 5' and 3' polynucleotide sequences have substantially the sequences selected from the group consisting of:
  • Genomic DNA was isolated from white blood cells of a subject with a family history of breast cancer. Dideoxy sequence analysis was performed following polymerase chain reaction amplification of segment L of exon 11.
  • Exon 11 of the BRCAl gene was subjected to direct dideoxy sequence analysis by asymmetric amplification using the polymerase chain reaction (PCR) to generate a single stranded product amplified from this DNA sample.
  • PCR polymerase chain reaction
  • Shuldiner, et al Handbook of Techniques in Endocrine Research, p. 457-486, DePablo, F., Scanes, C, eds., Academic Press, Inc., 1993. Fluorescent dye was attached for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer ® cat# 401628). DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) automated sequencer (Model 377). The software used for analysis of the resulting data was "Sequence Navigator" purchased through ABI.
  • Genomic DNA (100 nanograms) extracted from white blood cells of the subject was amplified in a final volume of 25 microliters containing 1 microliter (100 nanograms) genomic DNA, 2.5 microliters 10X PCR buffer (100 mM Tris, pH 8.3, 500 mM KC1, 1.2 mM MgCl 2 ), 2.5 microliters 10X dNTP mix (2 mM each nucleotide), 2.5 microliters forward primer (BRCA1- 11G-F, 10 micromolar solution), 2.5 microliters reverse primer (BRCA1- 11G-R, 10 micromolar solution), and 1 microliter Taq polymerase (5 units), and 13 microliters of water.
  • the PCR primers used to amplify segment G of exon 11 are as follows:
  • BRCA1-11G-F 5'-GGA AGTTAG CAC TCT AGG GA-3' SEQ ID NO:l
  • BRCA1-11G-R 5'-GCA GTG ATA TTA ACTGTC TGTA-3' SEQ ID NO:2
  • the primers were synthesized on a DNA /RNA Synthesizer Model 394®.
  • PCR products were purified using Qia-quick ® PCR purification kits (Qiagen ® , cat# 28104; Chatsworth, CA). Yield and purity of the PCR product determined spectrophotometrically at OD 26 o ° n a Beckman DU 650 spectrophotometer.
  • Fluorescent dye was attached to PCR products for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer ® cat# 401628).
  • 5'-CTT AAA GAA ACA AAG TC-3' (SEQ. ID. NO. 17), or 5'-CCT TAA AGA AAC AAA GT-3', (SEQ. ID. NO. 18) or with a sequence exhibiting the 2799delAA mutation such as 5'-CCT TAA AGA CAA AGT CC-3' (SEQ ID NO:9), or 5'-TCC TTA AAG ACA AAG TC-3' (SEQ ID NO:10).
  • the membranes were then washed twice in 2X SSPE and 0.05% SDS at
  • the 32 P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 2799delAA mutation, and not to those from "normal” individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (2799delAA) and negative (normal) controls were included in this analysis.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer.
  • Section G of exon 11 of the BRCA1 gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • the PCR primers used to amplify the suspected mutation are as follows:
  • BRCA1-11G-F 5'-CCT TAA AGA CAA AGT CC-3' SEQ ID NO:9
  • BRCA1-11G-R 5'-TCC TTA AAG ACA AAG TC-3' SEQ ID NO:10
  • the PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1.
  • the software used for analysis of the resulting data was "Sequence Navigator®" purchased through ABI.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section G of exon 11 of the
  • BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • BRCA1-11G-F 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l)
  • BRCA1-11G-R 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2)
  • PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1.
  • PCR products are purified using Qia-quick ® PCR purification kits (Qiagen, cat# 28104). DNA sequence analysis is performed as in EXAMPLE 1.
  • unlabeled PCR products from segment G of exon 11 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with 32 P labeled ATP oligonucleotides corresponding to either the normal sequence such as
  • 5'-CTT AAA GAA ACA AAG TC-3' (SEQ. ID. NO. 17), or 5'-CCT TAA AGA AAC AAA GT-3', (SEQ. ID. NO. 18) or with a sequence exhibiting the 2799delAA mutation such as
  • Segment L of exon 11 was amplified from genomic DNA by PCR as in EXAMPLE 1 using the following primers: BRCA1-11L-F: 5'-GTA ATA TTG GCA AAG GCA TCT-3' SEQ ID NO:3
  • the 32 P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 4158delAG mutation, and not to those from "normal” individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (4158delAG) and negative (normal) controls were included in this analysis.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section L of exon 11 of the
  • BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • BRCA1-11L-F 5'-ATG AAG AAA GGA ACG GG-3' SEQ ID NO:ll
  • BRCAl-llL-R 5'-GAT GAA GAA AGG AAC GG-3' SEQ ID NO:12 1.
  • the PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 4.
  • the software used for analysis of the resulting data was "Sequence Navigator ®" purchased through ABI.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer.
  • Section L of exon 11 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • the PCR primers used to amplify the suspected mutation are as follows:
  • BRCA1-11L-F 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3)
  • BRCAl-llL-R 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ ID NO:3)
  • unlabeled PCR products from segment L of exon 11 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with 32 P labeled ATP oligonucleotides corresponding to either the normal sequence
  • 5'-ATG AAG AAA GAG GAA CG-3' (SEQ. ID. NO: 19), or 5'-TGA AGA AAG AGG AAC GG -3', (SEQ. ID. NO:20) or with a sequence exhibiting the 4158delAG mutation such as 5'-ATG AAG AAA GGA ACG GG-3' (SEQ ID NO:ll), or
  • Exon 16 (where the mutation was found) was amplified from genomic DNA by PCR as in EXAMPLE 1 using the following primers:
  • BRCA1-16-F 5'-AAT TCT TAA CAG AGA CCA GAA C-3' SEQ ID NO:5
  • BRCA1-16-R 5'-AAA ACT CTT TCC AGA ATG TTG T-3' SEQ ID NO:6
  • Fluorescent dye was attached to PCR products for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer ® cat# 401628).
  • DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) Foster City, CA., automated sequencer (Model 377).
  • the software used for analysis of the resulting data was "Sequence Navigator ® " purchased through ABI.
  • 5'- AAC AGA AAG GGT CAA CA-3' (SEQ. ID. NO. 22) or with a sequence exhibiting the 5053delG mutation such as 5'-AAC AGA AAG GTC AAC AA-3' (SEQ ID NO:13), or
  • the 32 P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 5053delG mutation, and not to those from "normal” individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (5053delG) and negative (normal) controls were included in this analysis.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer.
  • Exon 16 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • the PCR primers used to amplify the suspected mutation are as follows: BRCA1-16-F: 5'-AAC AGA AAG GTC AAC AA-3' SEQ ID NO:13
  • BRCA1-16-R 5'-CAA CAG AAA GGT CAA CA-3' SEQ ID NO:14
  • the PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1. 2. Dideoxy Sequence Analysis
  • PCR products are purified using Qia-quick® PCR purification kits (Qiagen®, cat# 28104). Fluorescent dye is attached for automated sequencing using the Taq Dye Terminator® Kit (Perkin-Elmer cat# 401628). DNA sequencing is performed in accordance with the parameters specified in the Taq Dye Terminator® Kit, in both forward and reverse directions, on an Applied Biosystems, Inc ® (ABI) automated sequencer (Model 377).
  • Qia-quick® PCR purification kits Qiagen®, cat# 28104. Fluorescent dye is attached for automated sequencing using the Taq Dye Terminator® Kit (Perkin-Elmer cat# 401628). DNA sequencing is performed in accordance with the parameters specified in the Taq Dye Terminator® Kit, in both forward and reverse directions, on an Applied Biosystems, Inc ® (ABI) automated sequencer (Model 377).
  • the software used for analysis of the resulting data was "Sequence Navigator®" purchased through ABI.
  • nucleotide 5053 of the published BRCAl cDNA sequence can be found.
  • This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 1657.
  • This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein.
  • a finding of this mutation may be used either to design a program of gene therapy in a patient having a tumor, to characterize a tumor, or as a prognostic tool for those at risk of developing breast or ovarian cancer
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer.
  • Exon 16 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction
  • PCR PCR primers used to amplify the suspected mutation are as follows: BRCA1-16-F: 5'-AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5) BRCA1-16-R: 5'- AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6)
  • PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1. PCR products are purified using Qia-quick®
  • PCR purification kits Qiagen®, cat# 28104. Fluorescent dye is attached for automated sequencing using the Taq Dye Terminator® Kit (Perkin-Elmer cat# 401628). DNA sequencing is performed in both forward and reverse directions on an Applied Biosystems, Inc.® (ABI) automated sequencer (Model 377). The software used for analysis of the resulting data was
  • unlabeled PCR products from exon 16 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with 32 P labeled ATP oligonucleotides corresponding to either the normal sequence: 5'-ACA GAA AGG GTC AAC AA-3', (SEQ. ID. NO. 21), or 5'- AAC AGA AAG GGT CAA CA-3', (SEQ. ID. NO. 22) or with a sequence exhibiting the 5053delG mutation such as :
  • Segment A of exon 11 was amplified from genomic DNA by PCR amplification for approximately 35 cycles as described in EXAMPLE 1 using the following primers:
  • BRCA1-11A-F 5'-CCA CCT CCA AGG TGT ATC A-3' SEQ ID NO:7
  • BRCA1-11A-R 5'-TGT TAT GTT GGC TCC TTG CT-3' SEQ ID NO:8
  • the 32 P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 943insl0 mutation, and not to those from "normal” individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (943insl0) and negative (normal) controls were included in this analysis.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer.
  • Section A of exon 11 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • the PCR primers used to amplify the suspected mutation are as follows:
  • BRCA1-11A-F 5'-CCA TGT GGA GCC ATG TG-3' SEQ ID NO:15
  • BRCA1-11A-R 5'-GCC ATG TGG AGC CAT GT-3' SEQ ID NO: 16
  • the PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 10. 2. Dideoxy Sequence Analysis
  • the software used for analysis of the resulting data was "Sequence Navigator®" purchased through ABI.
  • Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section A of exon 11 of the
  • BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • BRCAl-11 A-F 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7)
  • BRC Al-11 A-R 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8)
  • PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 10.
  • PCR products are purified using Qia-quick® PCR purification kits (Qiagen ® , cat# 28104). Dideoxy sequence analysis is performed as described in EXAMPLE 10

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Abstract

Des mutations non encore reconnues étant apparues dans le gène BRCA1, l'invention concerne un procédé de diagnostic des individus susceptibles de développer un cancer du sein ou de l'ovaire. L'invention concerne également un outil complémentaire permettant de reconnaître le caractère de tumeurs.
PCT/US1997/013654 1996-08-05 1997-08-04 Mutation du gene de susceptibilite du cancer du sein et de l'ovaire WO1998005677A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006598A3 (fr) * 1997-08-04 1999-04-29 Oncormed Inc Determination d'alleles communs fonctionnels dans une population et applications de ceux-ci
US6951721B2 (en) 1996-02-12 2005-10-04 Gene Logic Inc. Method for determining the haplotype of a human BRCA1 gene

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5227292A (en) * 1990-07-12 1993-07-13 University Of Utah Neurofibromatosis type 1 gene

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5227292A (en) * 1990-07-12 1993-07-13 University Of Utah Neurofibromatosis type 1 gene

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUMAN MUTATION, 1996, Vol. 8, COUCH et al., "Mutations and Polymorphisms in the Familial Early-Onset Breast Cancer (BRCA1) Gene", pages 8-18. *
NATURE GENETICS, December 1994, Vol. 8, FRIEDMAN et al., "Confirmation of BRCA1 by Analysis of Germline Mutations Linked to Breast and Ovarian Cancer in Ten Families", pages 399-404. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951721B2 (en) 1996-02-12 2005-10-04 Gene Logic Inc. Method for determining the haplotype of a human BRCA1 gene
WO1999006598A3 (fr) * 1997-08-04 1999-04-29 Oncormed Inc Determination d'alleles communs fonctionnels dans une population et applications de ceux-ci

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