lrll:i;... t r. i:il, i::: i.:: ! -i'Y,, ' l..l l" l
`
`i':,1.,; 1'i1:i: l': ii: :.{: :+: r+ :+:
`i !".1 I i:} ,.ii:::,:.::,j.".ii,i
`.. .,:il
`i-.,
`i r.: ; ; j. !.... i"i
`
`,
`
`i.,,ii.,i i: {:.::i-.i
`if i.i.i. r...:j-i
`iri'ii. i..:i-r
`
`d1:".4.
`
`+: :i: :i: ;+: :i: :il: :i: i+: :1.:;. .i: :1:l.i
`,i. iii fi:l t::.: 'i i::' i:li:':'
`.ir-i tjl i...:j:i::' i ii::
`.i:i:i ll r:_: j. ii t.:ii::'
`.i. lr:; i:.i t-.: i i:r :"i;::,
`:i i:i fl i : 1 i::' ;"rP:'
`
`,* 3f/b
`.;
`'.i:'ji.r
`';;.,r;r
`
`t
`
`:
`
`i
`I
`
`:
`
`.?
`
`:r: :.i: r" . ,: ,,. : r' .. :, ,' i .j r. ; t-.
`, .. .... : .: .. i I i; !..i .i. i.l i_:;
`i.Ji:i:.i::i.i i:::f i ;::'i:i
`,/-
`/ va slril14,
`
`:
`
`. -
`
`'..-""-""-"'.--.."'-.
`
`1...i
`
`'! i::: i.,i /' ::, ! 1 !'
`j.- ! l- l r
`
`- uE-Elnhz
`
`;:::'l L-, Iitlt::i
`
`.ntfffuate
`
`.rur #iooa
`
`nrrm Qt
`d,Pit.
`
`EXAifilffi
`
`ffiJP$rm
`
`'{ PTO.1267 U.S. Department of Commerce
`11-92)
`PitenrandTrademad(Offioo
`t.i 1 DISCLAIMER LABET
`vF
`' Ap-olicalion No.
`
`per:aralgsTRoy
`
`.
`--=--
`om PIO438A
`(Rov.8/92)
`
`GeneDX 1009, pg. 1
`
`

`

`--r.r aool lgAT lOH *
`fSIg$ t c','...--
`-'' urnttlltlf
`
`(-n -f r.
`((' /1-?e'.
`ru/z-/ *?jr ,
`Li . 3t; '?b
`
`-\\$-\ V\f-
`
`'j.''
`
`1P.'
`
`20.
`
`21;
`'',1
`'2.
`
`ce
`
`.ffiF'.'
`
`GeneDX 1009, pg. 2
`
`

`

`INTERFERENCE SEARCHED
`
`-m——
`
`
`
`(RIGHT OUTSIDE) '
`
`§ G
`
`Kw
`
`eneDX 1009, pg. 3
`
`GeneDX 1009, pg. 3
`
`

`

`I
`
`.+A
`
`DATE
`
`,,
`
`+r &d -
`+ , ,
`' Y / d Y G .
`
`Staple Issue Slip her^
`
`ID NO.
`
`*,,'
`,
`
`+
`
`POSITION
`CLASSIFIER
`EXAMINER
`TYPIST
`VERIFIER
`CORPS CORR.
`SPEC. HAND
`FILE MAINT.
`DRAFTING
`
`455
`5-3-
`
`- 5 - .-"\ b
`6 -13- 4s
`
`*.-
`
`INDEX OF CLAIMS
`
`- ................................. Allowed
`
`J ............................... Rejected
`- (Through numberal) Canceled
`+ ................................. Restricted
`N ................................. Non-elected
`I ................................. Interlerence
`.................................
`A
`Appeal
`0 ................................. Objected
`
`(LEFT INSIDE)
`
`GeneDX 1009, pg. 4
`
`

`

`_ _ r + " l
`
`- - , -
`
`Staple Issue Slip Her:!
`-
`-
`-
`
`SYMBOLS
`J ......................... Rejected
`= ..................... Allowed
`(Through numberal) Canceled
`+ ......................... Aestrlctcrd
`N .......................... Non-elected
`......................
`I
`Interference
`A ........................ Appaal
`.......................
`0
`Objected
`
`( L E n INSIDE)
`
`a
`l x
`
`GeneDX 1009, pg. 5
`
`

`

`m , E OF THE INVENTION
`
`17q-LINJCl5D BREAST AND OVARIAN CANCER SUSCEPTIBILITY GENE
`
`ARSTRACT OF THE DISCLOSURE
`The present invention relates generally to the field of human genetics. Specifically, the present
`
`5
`
`invention relates to methods and materials used to isolate and detect a human breast and ovarian
`
`cancer predisposing gene (BRCAl), some mutant alleles of which cause susceptibility to cancer, in
`
`particular breast and ovarian cancer. More specifically, the invention relates to germline mutations
`in the BRCAl gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The
`
`10
`
`present invention further relates to somatic mutations in the BRCAl gene in human breast and
`ovarian cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer.
`
`Additionally, the invention relates to somatic mutations in the BRCAl gene in other human cancers
`
`and their use in the diagnosis and prognosis of human cancers. The invention also relates to the
`therapy of human cancers which have a mutation in the BRCAl gene, including gene therapy,
`
`15
`
`protein replacement therapy and protein mimetics. The invention further relates to the screening of
`
`drugs for cancer therapy. Finally, the invention relates to the screening of the BRCA1 gene for
`
`mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer.
`
`GeneDX 1009, pg. 6
`
`

`

`CANCER
`SUSCEPTIBILITY GENE
`- - .
`
`CROSS REFERENCE TO RELATED APP1,ICATIONS
`
`continuation-in-part of application Serial No. 081409,305 filed on 24 M
`
`ion-in-part of application Serial No. 081348,824 filed on 29 November 19
`
`which is a continuation-in-part of application Serial No. 081308,104 filed on 16 September 199
`
`is a continuation-in-part of application Serial No. 081300,266, filed on 2 September 19
`continuation-in-part of application Serial No. 081289,221, filed on 12 August 19941 4
`' / herein by reference.
`
`FIET ,D OF TU'E INVENTION
`
`15
`
`The present invention relates generally to the field of human genetics. Specifically, the present
`
`2 o
`
`invention relates to methods and materials used to isolate and detect a human breast and ovarian cancer
`
`predisposing gene (BRCAl), some mutant alleles of which cause susceptibility to cancer, in particular,
`
`breast and ovarian cancer. More specifically, the invention relates to gerrnline mutations in the BRCAl
`gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The present invention
`further relates to somatic mutations in the BRCAl gene in human breast and ovarian cancer and their
`use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention
`relates to somatic mutations in the BRCAl gene in other human cancers and their use in the diagnosis
`and prognosis of human cancers. The invention also relates to the therapy of human cancers which
`
`have a mutation in the BRCAl gene, including gene therapy, protein replacement therapy and protein
`
`25 mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the
`invention relates to the screening of the BRCAl gene for mutations, which are useful for diagnosing
`the predisposition to breast and ovarian cancer.
`
`The publications and other materials used herein to illuminate the background of the invention,
`
`and in particular, cases to provide additional details respecting the practice, are incorporated herein by
`
`3 0
`
`reference, and for convenience, are referenced by author and date in the following text and respectively
`grouped in the appended List of References.
`
`GeneDX 1009, pg. 7
`
`

`

`I
`I
`
`BACKGROUND OF THE INVENTION
`
`The genetics of cancer is complicated, involving multiple dominant, positive regulators of the
`
`transformed state (oncogenes) as well as multiple recessive, negative regulators (tumor suppressor
`
`5
`
`genes). Over one hundred oncogenes have been characterized. Fewer than a dozen tumor suppressor
`
`genes have been identified, but the number is expected to increase beyond fifty (Knudson, 1993).
`
`The involvement of so many genes underscores the complexity of the growth ,control
`
`mechanisms that operate in cells to maintain the integrity of normal tissue. This complexity is manifest
`
`in another way. So far, no single gene has been shown to participate in the development of all, or even
`
`1 0
`
`the majority of human cancers. The most common oncogenic mutations are in the H-ras gene, found in
`
`10-1 5% of all solid tumors (Anderson et al., 1992). The most frequently mutated tumor suppressor
`
`genes are the TP53 gene, homozygously deleted in roughly 50% of all tumors, and CDKN2, which was
`homozygously deleted in 46% of tumor cell lines examined (Kamb et al., 1994). Without a target that
`
`is common to all transformed cells, the dream of a "magic bullet" that can destroy or revert cancer cells
`
`15 while leaving noimal tissue unharmed is improbable. The hope for a new generation of specifically
`
`targeted antitumor drugs may rest on the ability to identi@ tumor suppressor genes or oncogenes that
`
`play general roles in control of cell division.
`
`The tumor suppressor genes which have been cloned and characterized influence susceptibility
`
`to: 1) Retinoblastoma (RBI); 2) Wilms' tumor (WT1); 3) Li-Fraumeni (TP53); 4) Familial adeno-
`2 0 matous polyposis (APC); 5) Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7)
`von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); and 9)
`
`Melanoma (CDKN2).
`
`Tumor suppressor loci that have been mapped genetically but not yet isolated include genes for:
`
`Multiple endocrine neoplasia type 1 (MEN1); Lynch cancer family syndrome 2 (LCFS2);
`
`25 Neuroblastoma (NB); Basal cell nevus syndrome (BCNS); Beckwith-Wiedernann syndrome (BWS);
`Renal cell carcinoma (RCC); Tuberous sclerosis 1 (TSC1); and Tuberous sclerosis 2 (TSC2). The
`
`tumor suppressor genes that have been characterized to date encode products with similarities to a
`variety of protein types, including DNA binding proteins (WTl), ancillary transcription regulators
`
`(RBI), GTPase activating proteins or GAPS (NFl), cytoskeletal components (NF2), membrane bound
`
`3 0
`
`receptor kinases (MEN2A), cell cycle regulators (CDKN2) and others with no obvious similarity to
`known proteins (APC and VHL).
`
`GeneDX 1009, pg. 8
`
`

`

`shown to be lost or mutated in some sporadic tumors. This result suggests that regions of chromosomal
`
`aberration may signify the position of important hunor suppressor genes involved both in genetic
`predisposition to cancer and in sporadic cancer.
`One of the hallmarks of several tumor suppressor genes characterized to date is that they are
`
`5
`
`deleted at high frequency in certain tumor types. The deletions often involve loss of a single allele, a
`so-called loss of heterozygosity (LOH), but may also involve homozygous deletion of both alleles. For
`LOH, the remaining allele is presumed to be nonfunctional, either because of a preexisting inherited
`
`mutation, or because of a secondary sporadic mutation.
`Breast cancer is one of the most significant diseases that affects women. At the current rate,
`
`10
`
`American women have a 1 in 8 risk of developing breast cancer by age 95 (American Cancer Society,
`
`1992). Treatment of breast cancer at later stages is often futile and disfiguring, making early detection
`
`a high priority in medical management of the disease. Ovarian cancer, although less frequent than
`
`breast cancer is often rapidly fatal and is the fourth most common cause of cancer mortality in
`15 American women. Genetic factors contribute to an ill-defined proportion of breast cancer incidence,
`
`2 0
`
`25
`
`estimated to be about 5% of all cases but approximately 25% of cases diagnosed before age 40 (Claus
`
`et al., 1991). Breast cancer has been subdivided into two types, early-age onset and late-age onset,
`
`based on an inflection in the age-specific incidence curve around age 50. Mutation of one gene,
`
`BRCA1, is thought to account for approximately 45% of familial breast cancer, but at least 80% of
`
`families with both breast and ovarian cancer (Easton et al., 1993).
`Intense efforts to isolate the BRCAl gene have proceeded since it was first mapped in 1990 (Hall
`et al., 1990; Narod et al., 199 1). A second locus, BRCA2, has recently been mapped to chromosome
`13q (Wooster et al., 1994) and appears to account for a proportion of early-onset breast cancer roughly
`
`equal to BRCAl, but confers a lower risk of ovarian cancer. The remaining susceptibility to early-
`
`onset breast cancer is divided between as yet unmapped genes for familial cancer, and rarer germline
`mutations in genes such as TP53 (Malkin et al., 1990). It has also been suggested that heterozygote
`carriers for defective foms of the Ataxia-Telangectasia gene are at higher risk for breast cancer (Swift
`et al., 1976; Swift et al., 1991). Late-age onset breast cancer is also often familial although the risks in
`relatives are not as high as those for early-onset breast cancer (Cannon-Albright et al., 1994; Mettlin et
`
`GeneDX 1009, pg. 9
`
`

`

`Breast cancer has long been recognized to be, in part, a familial disease (Anderson, 1972).
`
`Numerous investigators have examined the evidence for genetic inheritance and concluded that the data
`
`5
`
`are most consistent with dominant inheritance for a major susceptibility locus or loci (Bishop and
`Gardner, 1980; Go et al., 1983; Willams and Anderson, 1984; Bishop et al., 1988; Newrnan et al.,
`1988; Claus et al., 1991). Recent results demonstrate that at least three loci exist which convey
`susceptibility to breast cancer as well as other cancers. n e s e loci are the TP53 locus on chromosome
`17p (Mallcin et al., 1990), a 17q-linked susceptibility locus known as BRCAl (Hall et al., 1990), and
`
`one or more loci responsible for the unmapped residual. Hall et al. (1990) indicated that the inherited
`
`breast cancer susceptibility in kindreds with early age onset is linked to chromosome 17q21; although
`subsequent studies by this group using a more appropriate genetic model partially refuted the limitation
`
`1 o
`
`to early onset breast cancer (Margaritte et al., 1992).
`Most strategies for cloning the l7q-linked breast cancer predisposing gene (BRCAl) require
`
`precise genetic localization studies. The simplest model for the functional role of BRCAl holds that
`
`alleles of BRCAl that predispose to cancer are recessive to wild type alleles; that is, cells that contain
`
`15
`
`at least one wild type BRCAl allele are not cancerous, However, cells that contain one wild type
`
`BRCAl allele and one predisposing allele may occasionally suffer loss of the wild type allele either by
`random mutation or by chromosome loss during cell division (nondisjunction). All the progeny of such
`a mutant cell lack the wild type function of BRCAl and may develop into tumors, According to this
`
`model, predisposing alleles of BRCAl are recessive, yet susceptibility to cancer is inherited in a
`
`2 0
`
`dominant fashion: women who possess one predisposing allele (and one wild type allele) risk
`
`developing cancer, because their mammary epithelial cells may spontaneously lose the wild type
`
`BRCAl allele. This model applies to a group of cancer susceptibility loci known as tumor suppressors
`or antioncogenes, a class of genes that includes the retinoblastoma gene and neurofibromatosis gene.
`
`2 5
`
`By inference this model may also explain the BRCAl function, as has recently been suggested (Smith
`et al., 1992).
`A second possibility is that BRCAl predisposing alleles are truly dominant; that is, a wild type
`allele of BRCAl cannot overcome the tumor forming role of the predisposing allele. Thus, a cell that
`
`carries both wild type and mutant alleles would not necessarily lose the wild type copy of BRCAl
`
`before giving rise to malignant cells. Instead, mammary cells in predisposed individuals would
`undergo some other stochastic change(s) leading to cancer.
`
`3 0
`
`GeneDX 1009, pg. 10
`
`

`

`If BRCAl predisposing alleles are recessive, the BRCAl gene is expected to be expressed in
`
`normal mammary tissue but not functionally expressed in mammary hunors. In contrast, if BRCAl
`
`predisposing alleles are dominant, the wild type BRCAl gene may or may not be expressed in normal
`
`mammary tissue. However, the predisposing allele will likely be expressed in breast tumor cells.
`
`The 17q linkage of BRCAl was independently confirmed in three of five kindreds with both
`
`breast cancer and ovarian cancer (Narod et al., 1991). These studies claimed to localize the gene within
`a very large region, 15 centiMorgans (cM), or approximately 15 million base pairs, to either side of the
`linked marker pCMM86 (D17S74). However, attempts to defme the region further by genetic studies,
`using markers surrounding pCMMS6, proved unsuccessful. Subsequent studies indicated that the gene
`was considerably more proximal (Easton et al., 1993) and that the original analysis was flawed
`(Margaritte et al., 1992). Hall et al., (1992) recently localized the BRCAl gene to an approximately 8
`
`cM interval (approximately 8 million base pairs) bounded by Mfdl5 (D17S250) on the proximal side
`
`and the human GIP gene on the distal side. A slightly narrower interval for the BRCAl locus, based on
`publicly available data, was agreed upon at the Chromosome 17 workshop in March of 1992 (Fain,
`
`1992). The size of these regions and the uncertainty associated with them has made it exceedingly
`
`dificult to design and implement physical mapping andlor cloning strategies for isolating the BRCAl
`
`gene.
`
`Identification of a breast cancer susceptibility locus would permit the early detection of
`susceptible individuals and greatly increase our ability to understand the initial steps which lead to
`cancer. As susceptibility loci arc oftcn altcred during tumor progression, cloning these genes could
`
`also be important in the development of better diagnostic and prognostic products, as well as better
`
`cancer therapies.
`
`P
`
`RY OF THE INVENTION
`
`2 0
`
`25
`
`The present invention relates generally to the field of human genetics. Specifically, the present
`
`invention relates to methods and materials used to isolate and detect a human breast cancer
`
`predisposing gene (BRCAl), some alleles of which cause susceptibility to cancer, in particular breast
`and ovarian cancer. More specifically, the present invention relates to germline mutations in the
`
`3 0
`
`BRCAl gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The
`invention further relates to somatic mutations in the BRCAl gene in human breast cancer and their use
`
`GeneDX 1009, pg. 11
`
`

`

`in the diagnosis and prognosis of hurnan breast and ovarian cancer. Additionally, the invention relates
`
`to somatic mutations in the BRCAl gene in other hurnan cancers and their use in the diagnosis and
`prognosis of human cancers. The invention also relates to the therapy of human cancers which have a
`
`5
`
`mutation in the BRCAl gene, including gene therapy, protein replacement therapy and protein
`rnimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the
`invention relates to the screening of the BRCAl gene for mutations, which are useful for diagnosing
`the predisposition to breast and ovarian cancer.
`
`I
`
` BRIEF-^
`
`10
`
`15
`
`2 o
`
`2 5
`
`Figure 1 is a diagram showing the order of loci neighboring BRCAl as determined by the
`chromosome 17 workshop. Figure 1 is reproduced from Fain, 1992.
`Figure 2 is a schematic rnap of YACs which define part of Mfdl5-Mfdl88 region.
`Figure 3 is a schematic map of STSs, Pls and BACs in the BRCAl region.
`Figure 4 is a schematic map of human chromosome 17. The pertinent region containing BRCAl
`
`is expanded to indicate the relative positions of two previously identified genes, CA125 and RNU2,
`
`BRCAl spans the marker Dl 7S855.
`Figure 5 shows alignment of the BRCAl zinc-finger domain with 3 other zinc-finger domains
`that scored highest in a Smith-Waterman alignment. RPTl encodes a protein that appears to be a
`negative regulator of the IL-2 receptor in mouse. RINl encodes a DNA-binding protein that includes a
`FWG-finger motif related to the zinc-finger. WPl encodes a putative transcription factor that is the
`
`N-terminal domain of the RET oncogene product. The bottom line contains the C3HC4 consensus
`
`zinc-finger sequence showing the positions of cysteines and one histidine that form the zinc ion binding
`pocket.
`Figure 6 is a diagram of BRCAl mRNA showing the locations of introns and the variants of
`
`BRCAl mRNA produced by alternative splicing. Intron locations are shown by dark triangles and the
`exons are numbered below the line representing the D N A . The top cDNA is the composite used to
`
`generate the peptide sequence o f BRCAl. Alternative forms identified as cDNA clones or hybrid
`selection clones are shown below.
`
`3 0
`
`Figure 7 shows the tissue expression pattern of BRCAI, The blot was obtained from Clontech
`
`and contaii~s RNA from thc indicated tissucs. I-Iybridi=tion conditions wcre as rccommendcd by the
`
`I
`
`t
`
`GeneDX 1009, pg. 12
`
`

`

`manufacturer using a probe consisting of nucleotide positions 363 1 to 3930 of BRCAl. Note that both
`
`breast and ovary are heterogeneous tissues and the percentage of relevant epithelial cells can be
`
`variable. Molecular weight standards are in kilobases.
`Figure 8 is a diagram of the 5' untranslated region plus the beginning of the translated region of
`BRCAl showing the locations of introns and the variants of BRCAl mRNA produced by alternative
`
`splicing. Intron locations are shown by broken dashed lines. Six alternate splice forms are shown.
`
`Figure 9A shows a nonsense mutation in Kindred 2082. P indicates the person originally
`screened, b and c are haplotype carriers, a, d, e, f, and g do not carry the BRCAl haplotype. The C to T
`mutation results in a stop codon and creates a site for the restriction enzyme AvrII. PCR amplification
`products are cut with this enzyme. The carriers are heterozygous for the site and therefore show three
`bands. Non-carriers remain uncut.
`Figure 9B shows a mutation and cosegregation analysis in BRCAl kindreds. Carrier individuals
`are represented as filled circles and squares in the pedigree diagrams. Frarneshift mutation in Kindred
`1910. The first three lanes are control, noncarrier samples. Lanes labeled 1-3 contain sequences fiom
`
`carrier individuals. Lane 4 contains DNA from a kindred member who does not carry the BRCAl
`
`mutation. The diamond is used to prevent identification of the kindred. The frameshift resulting fiom
`
`the additional C is apparent in lanes labeled 1,2, and 3.
`
`Figure 9C shows a mutation and cosegregation analysis in BRCAl kindreds. Carrier individuals
`
`are represented as filled circles and squares in the pedigree diagrams. Inferred regulatory mutation in
`Kindred 2035. AS0 analysis of carriers and noncarriers of 2 different polymorphisms (PM1 and PM7)
`which were examined for heterozygosity in the germline and compared to the heterozygosity of
`
`lymphocyte rnRNA. The top 2 rows of each panel contain PCR products amplified from genomic
`
`DNA and the bottom 2 rows contain PCR products amplified fiom cDNA. "A" and "G" are the two
`
`alleles detected by the ASO. The dark spots indicate that a particular allele is present in the sample.
`The first three lanes of PM7 represent the three genotypes in the general population.
`Figures 10A-1OH show genomic sequence of BRCAl. The lower case letters denote intron
`
`sequence while the upper case letters denote exon sequence. Indefinite intervals within introns are
`
`designated with vwwwwww. Known polymorphic sites are shown as underlined and boldface
`
`tY Pe.
`
`GeneDX 1009, pg. 13
`
`

`

`-8-
`
`AILED DESCRIPTION OF THE INVENTION
`
`The present invention relates generally to the field of human genetics. Specifically, the present
`
`invention relates to methods and materials used to isolate and detect a human breast cancer
`predisposing gene (BRCAl), some alleles of which cause susceptibility to cancer, in particular breast
`
`5
`
`and ovarian cancer. More specifically, the present invention relates to germline mutations in the
`BRCAl gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The
`
`invention further relates to somatic mutations in the BRCAl gene in human breast cancer and their use
`
`in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates
`10 to somatic mutations in the BRCAl gene in other human cancers and their use in the diagnosis and
`
`prognosis of human cancers. The invention also relates to the therapy of human cancers which have a
`
`mutation in the BRCAl gene, including gene therapy, protein replacement therapy and protein
`
`mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the
`
`invention relates to the screening of the BRCAl gene for mutations, which are useful for diagnosing
`
`15
`
`the predisposition to breast and ovarian cancer.
`
`The present invention provides an isolated polynucleotide comprising all, or a portion of the
`BRCAl locus or of a mutated BRCAl locus, preferably at least eight bases and not more than about
`
`100 kb in length. Such polynucIeotides may be antisense polynucleotides. The present invention also
`provides a recombinant construct comprising such an isolated polynucleotide, for example, a
`
`2 0
`
`recombinant construct suitable for expression in a transformed host cell.
`
`Also provided by the present invention are methods of detecting a polynucleotide comprising a
`
`portion of the BRCAl locus or its expression product in an analyte. Such methods may further
`comprise the step of amplifying the portion of the BRCAl locus, and may fiuther include a step of
`
`providing a set of polynucleotides which are primers for amplification of said portion of the BRCAl
`
`25
`
`locus. The rnethcd is useful for either diagnosis of the predisposition to cancer or the diagnosis or
`prognosis of cancer.
`
`The present invention also provides isolated antibodies, preferably monoclonal antibodies, which
`specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by
`
`the BRCAl locus.
`
`GeneDX 1009, pg. 14
`
`

`

`The present invention also provides kits for detecting in an analyte a polynucleotide comprising a
`
`portion of the BRCAl locus, the kits comprising a polynucleotide complementary to the portion of the
`
`BRCAl locus packaged in a suitable container, and instructions for its use.
`
`The present invention further provides methods of preparing a polynucleotide comprising
`polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the
`
`BRCAl locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a
`
`sequence comprising at least five amino acids encoded within the BRCAl locus.
`
`The present invention further provides methods of screening the BRCAl gene to identify
`
`mutations. Such methods may further comprise the step of amplifying a portion of the BRCAl locus,
`and may further include a step of providing a set of polynucleotides which are primers for amplification
`
`of said portion of the BRCAl locus. The method is useful for identifying mutations for use in either
`
`diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.
`The present invention further provides methods of screening suspected BRCAl mutant alleles to
`identify mutations in the BRCAl gene.
`
`In addition, the present invention provides methods of screening drugs for cancer therapy to
`
`identify suitable drugs for restoring BRCA1 gene product function.
`
`Finally, the present invention provides the means necessary for production of gene-based
`
`therapies directed at cancer cells. These therapeutic agents may take the form of polynucleotides
`
`comprising all or a portion of the BRCAl locus placed in appropriate vectors or delivered to target cells
`
`in more direct ways such that the function of the BRCAl protein is reconstituted. Therapeutic agents
`
`may also take the fonn of polypeptides based on either a portion of, or the entire protein sequence of
`BRCA1. These may functionally replace the activity of BRCAl in vivo.
`
`It is a discovery of the present invention that the BRCAl locus which predisposes individuals to
`
`breast cancer and ovarian cancer, is a gene encoding a BRCAl protein, which has been found to have
`
`no significant homology with known protein or DNA sequences. This gene is termed BRCAl herein.
`It is a discovery of the present invention that mutations in the BRCAl locus in the gerrnline are
`indicative of a predisposition to breast cancer and ovarian cancer. Finally, it is a discovery of the
`present invention that somatic mutations in the BRCAl locus are also associated with breast cancer,
`
`ovarian cancer and other cancers, which represents an indicator of these cancers or of the prognosis of
`
`these cancers. The mutational events of the BRCAl locus can involve deletions, insertions and point
`
`GeneDX 1009, pg. 15
`
`

`

`Starting fiom a region on the long arm of human chromosome 17 of the hurnan genome, 17q,
`
`which has a size estimated at about 8 million base pairs, a region which contains a genetic locus,
`
`BRCAl, which causes susceptibility to cancer, including breast and ovarian cancer, has been identified.
`
`The region containing the BRCAl locus was identified using a variety of genetic techniques.
`
`Genetic mapping techniques initially defined the BRCAl region in terms of recombination with genetic
`
`markers. Based upon studies of large extended families ("kindreds") with multiple cases of breast
`
`cancer (and ovarian cancer cases in some kindreds), a chromosomal region has been pinpointed that
`
`contains the BRCAl gene as well as other putative susceptibility alleles in the BRCAl locus. Two
`
`meiotic breakpoints have been discovered on the distal side of the BRCAl locus which are expressed
`
`as recombinants between genetic markers and the disease, and one recombinant on the proximal side of
`
`the BRCAl locus. Thus, a region which contains the BRCAl locus is physically bounded by these
`
`markers.
`
`The use of the genetic markers provided by this invention allowed the identification of clones
`which cover the region from a hurnan yeast artificial chromosome (YAC) or a human bacterial artificial
`chromosome (BAC) library. It also allowed for the identification and preparation of more easily
`
`manipulated cosmid, P1 and BAC clones fiom this region and the construction of a contig fiom a
`
`subset of the clones. These cosmids, Pls, YACs and BACs provide the basis for cloning the BRCAl
`
`locus and provide the basis for developing reagents effective, for example, in the diagnosis and
`
`treatment of breast andlor ovarian cancer. The BRCAl gene and other potential susceptibility genes
`
`have been isolated from this region. The isolation was done using software trapping (a computational
`
`method for identifying sequences likely to contain coding exons, from contiguous or discontinuous
`
`genomic DNA sequences), hybrid selection techniques and direct screening, with whole or partial
`
`cDNA inserts from cosmids, Pls and BACs, in the region to screen cDNA libraries. These methods
`
`were used to obtain sequences of loci expressed in breast and other tissue. These candidate loci were
`
`analyzed to identi@ sequences which confer cancer susceptibility. We have discovered that there are
`mutations in the coding sequence of the BRCAl locus in kindreds which are responsible for the 17q-
`
`linked cancer susceptibility known as BRCAl. This gene was not known to be in this region. The
`
`present invention not only facilitates the early detection of certain cancers, so vital to patient survival,
`
`but also permits the detection of susceptible individuals before they develop cancer.
`
`GeneDX 1009, pg. 16
`
`

`

`Population Resources
`
`Large, well-documented Utah kindreds are especially important in providing good resources for
`
`human genetic studies. Each large kindred independently provides the power to detect whether a
`
`BRCAl susceptibility allele is segregating in that family. Recombinants informative for localization
`and isolation of the BRCAl locus could be obtained only from kindreds large enough to confirm the
`
`5
`
`10
`
`15
`
`2 0
`
`25
`
`presence of a susceptibility allele. Large sibships are especially important for studying breast cancer,
`
`since penetrance of the BRCAl susceptibility allele is reduced both by age and sex, making
`informative sibships difficult to find. Furthermore, large sibships are essential for constructing
`haplotypes of deceased individuals by inference fiom the haplotypes of their close relatives.
`While other populations may also provide beneficial information, such studies generally require
`
`much greater effort, and the families are usually much smaller and thus less informative. Utah's age-
`
`adjusted breast cancer incidence is 20% lower than the average U.S. rate. The lower incidence in Utah
`
`is probably due largely to an early age at first pregnancy, increasing the probability that cases found in
`
`Utah kindreds carry a genetic predisposition.
`
`Genetic Mapping
`Given a set of informative families, genetic markers are essential for linking a disease to a region
`
`of a chromosome. Such markers include restriction fragment length polymorphisms (RFLPs) (Botstein
`et aL, 1980), markers with a variable number of tandem repeats (VNTRs) (Jeffieys et al., 1985;
`Nakarnura et al., 1987), and an abundant class of DNA polymorphisms based on short tandem repeats
`(STRs), especially repeats of CpA (Weber and May, 1989; Litt et al., 1989). To generate a genetic map,
`one selects potential genetic markers and tests them using DNA extracted from members of the
`
`kindreds being studied.
`
`Genetic markers useful in searching for a genetic locus associated with a disease can be selected
`
`on an ad hoc basis, by densely covering a specific chromosome, or by detailed analysis of a specific
`region of a chromosome. A preferred method for selecting genetic markers linked with a disease
`involves