WO1998023779A1 - Les promoteurs des genes brca1 et 1a1.3b sont des elements paralleles d'une duplication genomique au niveau de 17q21 - Google Patents

Les promoteurs des genes brca1 et 1a1.3b sont des elements paralleles d'une duplication genomique au niveau de 17q21 Download PDF

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WO1998023779A1
WO1998023779A1 PCT/US1997/021358 US9721358W WO9823779A1 WO 1998023779 A1 WO1998023779 A1 WO 1998023779A1 US 9721358 W US9721358 W US 9721358W WO 9823779 A1 WO9823779 A1 WO 9823779A1
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brcal
gene
seq
seconds
polymoφhism
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David F. Barker
Xudong Liu
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University Of Utah Research Foundation
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    • 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
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    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • 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
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    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates generally to the field of human genetics. Specifically, the present invention relates to a gene, named LBRCA1 (which stands for "Like BRCAl”), which is very similar to a human breast and ovarian cancer predisposing gene (BRCAl), some mutant alleles of which cause susceptibility to cancer.
  • the invention also relates to a gene called 1A1.3B and a very similar gene named L1A1.3B (for Like 1A1.3B).
  • L1A1.3B is located extremely close to BRCAl in a head to head configuration while LBRCA1 and 1A1.3B are similarly located very close to each other also in a head to head arrangement, wherein genes that have 5' ends located immediately adjacent to one another are said to be "head-to-head”.
  • the BRCA1/L1A1.3B and LBRCA1/1A1.3B regions are a result of gene duplication. Knowledge of the LBRCA1 sequence is important for the analysis of BRCAl for mutations because the very high similarity between the two genes could lead to problems when trying to analyze BRCAl. Extensive testing of persons for mutations in BRCAl is expected to begin very soon.
  • the LBRCA1 and L1A1.3B contain promoter regions similar to the promoters for BRCAl and 1A1.3B. These additional promoters, which are in close proximity to the BRCAl and 1A1.3B genes, may affect transcription of these latter genes.
  • a further aspect of the present invention is that the knowledge of the chromosomal arrangement of these genes and the fact that there has been a gene duplication, is useful in looking for mutations, other than mutations directly within BRCAl, which could affect proper transcription of BRCAl and may be responsible for breast or ovarian cancer.
  • Another aspect of the invention is that polymorphisms in or near LBRCA1 and L1A1.3B have been found and these are useful in tracking the chromosomal arrangement of these genes as well as BRCAl and 1A1.3B to determine whether rearrangement has occurred.
  • oncogenes dominant, positive regulators of the transformed state
  • tumor suppressor genes multiple recessive, negative regulators (tumor suppressor 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 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 adenomatous polyposis (APC); 5) Neurofibromatosis type 1 (NFl); 6) Neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); and 9) Melanoma (CDKN2).
  • RBI Retinoblastoma
  • WT1 Wilms' tumor
  • TP53 Li-Fraumeni
  • APC Familial adenomatous polyposis
  • NFl Neurofibromatosis type 1
  • NF2 Neurofibromatosis type 2
  • VHL von Hippel-Lindau syndrome
  • MEN2A Multiple endocrine neo
  • 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); Neuroblastoma (NB); Basal cell nevus syndrome (BCNS); Beckwith-Wiedemann syndrome (BWS); Renal cell carcinoma (RCC); Tuberous sclerosis 1 (TSC1); and Tuberous sclerosis 2 (TSC2).
  • MEN1 Multiple endocrine neoplasia type 1
  • LCFS2 Lynch cancer family syndrome 2
  • NB Neuroblastoma
  • BCNS Basal cell nevus syndrome
  • BCNS Basal cell nevus syndrome
  • BWS Beckwith-Wiedemann syndrome
  • RRCC Renal cell carcinoma
  • TSC1 Tuberous sclerosis 1
  • TSC2 Tuberous sclerosis 2
  • the tumor suppressor genes that have been characterized to date encode products with similarities to a variety of protein types, including DNA binding proteins (WT1), ancillary transcription regulators (RBI), GTPase activating proteins or GAPs (NFl), cytoskeletal components (NF2), membrane bound receptor kinases (MEN2A), cell cycle regulators (CDKN2) and others with no obvious similarity to known proteins (APC and VHL).
  • WT1 DNA binding proteins
  • RBI ancillary transcription regulators
  • NFl GTPase activating proteins or GAPs
  • NF2 cytoskeletal components
  • MEN2A membrane bound receptor kinases
  • CDKN2 cell cycle regulators
  • APC and VHL cell cycle regulators
  • the tumor suppressor gene originally identified through genetic studies has been shown to be lost or mutated in some sporadic tumors. This result suggests that regions of chromosomal aberration may signify the position of important tumor suppressor genes involved both in genetic predisposition to cancer and in sporadic cancer.
  • LOH loss of heterozygosity
  • BRCA2 BRCA2A gene was 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).
  • loci are the TP53 locus on chromosome 17p (Malkin 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.
  • BRCAl and BRCA2 genes have recently been identified. These are located on chromosomes 17q and 13q, respectively. 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 to early onset breast cancer (Margaritte et al, 1992).
  • 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 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.
  • predisposing alleles of BRCAl are recessive, yet susceptibility to cancer is inherited in a 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. By inference this model may also explain the BRCAl function, as has recently been suggested (Smith et al, 1992).
  • BRCAl predisposing alleles are truly dominant; that is, a wild type allele of BRCAl cannot overcome the tumor forming role of the predisposing allele.
  • 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.
  • BRCAl predisposing alleles are recessive, the BRCAl gene is expected to be expressed in normal mammary tissue but not functionally expressed in mammary tumors. 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.
  • BRCAl is located very near to LI A1.3B, which is a partial duplication of the 1A1.3B gene, which is located very near to LBRCA1.
  • the L1A1.3B lies head to head within 250 base pairs of BRCAl.
  • the overlapping of regulatory regions for the two genes may be of importance in coordinate control of the two genes.
  • the presence of a duplication containing all or part of BRCAl and 1A1.3B suggests that recombination events or other homology-mediated genetic rearrangements, occurring somatically or as heritable changes, could result in altered expression or inactivation of genes located within or close to the duplicated segment, including, but not limited to, the BRCAl and 1A1.3B genes.
  • polymorphisms have been found in LBRCA 1 and the BRCAl promoter region. These will be useful in characterizing possible mutations in LBRCA 1 and will also be useful for "diagnosing" chromosomal rearrangements involving LBRCA1. This is important because with other genes it has been shown that duplication of a segment of human DNA results in a predisposition to genomic rearrangements that are associated with disease. Such a mechanism may also occur with BRCAl and such rearrangements may be responsible for causing cancer rather than, e.g., a missense or nonsense mutation within the gene. This mechanism may be important either to cause heritable defects or to create gene defects during the somatic growth of cells that carry no inherited defect.
  • the present invention relates generally to the field of human genetics. Specifically, the present invention relates to the duplication of a portion of human chromosome 17q containing the breast cancer gene BRCAl.
  • the invention relates to the chromosomal arrangement and sequence similarities of BRCA1-L1A1.3B and LBRCA- 1A1.3B.
  • This invention further relates to LBRCA1 polymorphisms and BRCAl promoter region polymorphisms that are useful in analyzing whether genomic rearrangements have occurred and the usefulness of this in the diagnosis and prognosis of human breast and ovarian cancer.
  • Figure 1A is a summary of chromosomal localization of specific PCR products and restriction mapping of genomic clones for the BRCAl promoter and its cognate.
  • the regions of chromosome 17 contained in the rodent human hybrids ND-1 and MH-41 are indicated by solid horizontal lines (vanTuinen et al., 1987).
  • the inferred relative locations and sizes of the genomic EcoRI fragments corresponding to those in CH40 clones 10A and 16C are shown.
  • a conserved central EcoRI site is located between the most 5' exons of the head-to-head gene arrangements and is marked by a double-thick hash.
  • the 16C clone was first identified as containing BRCAl -specific sequences because it showed greater hybridization to the oligonucleotide 1007-5 than the 10A clone, indicated here by heavy and light arrows, as described.
  • Figure IB shows an STS analysis of YAC and PI clones previously mapped to the BRCAl region (Albertsen et al., 1994, Neuhausen et al., 1994) and the 10A and 16C clones described elsewhere in the specification.
  • PCR primer combinations are as described in Table 1 or elsewhere in the disclosure.
  • Figure 2 is a summary of features of the BRCAl and 1A1.3B promoter region sequences. Known exons of these two genes are indicated as solid boxes and the corresponding regions of the putative cognate genes with the same apparent intron-exon boundaries are indicated as checkered boxes. The largest segments unique to each sequence are indicated as open triangles, with the length indicated in bp.
  • the EcoRI site marked is the central EcoRI site noted in Figure 1.
  • the position of the BRCAl major translation product start site is indicated in exon 2 and a similar indication is shown for a possible translation start site in the corresponding segment of LBRCA 1.
  • the approximate locations of oligonucleotide primer sequences described in Table 1 are shown.
  • the open boxes at positions 150 and 525 on the basepair scale represent polymorphisms detected in the BRCAl promoter.
  • the narrow box indicates a base difference, C or T
  • the wide box indicates a trinucleotide difference, AAC or AACAAC.
  • SEQ ID NO:l shown in the Sequence Listing as SEQ ID NO:l
  • these correspond to nucleotide positions 612, which is C in U37574 and 980-982 where U37574 contains a single trinucleotide element.
  • These polymorphisms are in apparent strong linkage disequilibrium, with the C/AAC haplotype having a frequency of 0.65 on 190 tested chromosomes.
  • Figure 3 is a dot-plot comparison of U37574 with the sequence derived from genomic clone 10A (Genbank accession U72483 (shown in the Sequence Listing as SEQ ID NO:2)). For this comparison a window size of 15 and a match criterion of 12 were used. The positions of the known and comparable exon structures for BRCAl, LBRCA1, 1 Al .3B and LI A1.3B are marked along the axes in the same format as shown in Figure 2. The significant gaps representing the largest differences between the sequences discussed in the text are indicated.
  • the present invention relates generally to the field of human genetics. Specifically, the present invention relates to a partial duplication of a human breast cancer predisposing gene (BRCAl), and some polymorphic allelic forms which can be useful in tracking the chromosomal arrangement of BRCAl.
  • BRCAl human breast cancer predisposing gene
  • the invention also relates to the fact that there can be mutations in the LBRCA 1 and L1A1.3B promoter regions that can affect transcription of the BRCAl and 1A1.3B genes.
  • BRCAl Mutations in the BRCAl gene are associated with a highly increased risk of breast or ovarian cancer development, and inheritance of defective forms of this gene may account for approximately 5% of breast cancer cases. Altered expression or effective loss of function of BRCAl is likely to be important in sporadic breast and ovarian tumors as well (Chen et al., 1995; Holt et al., 1996). Although a complete genomic structure of BRCAl ismot yet available, a complete coding region cDNA sequence of BRCAl has been reported (Miki et al., 1994).
  • the cDNA structure was further elucidated in a report characterizing the promoter region of BRCAl and describing the alternative use of exons la or lb in different tissue types (Xu et al., 1995).
  • An additional complexity of BRCAl transcription was noted by Brown et al. (1994) who provided evidence that the 1A1.3B gene identified by Campbell et al. (1994) and believed to encode the CA125 ovarian cancer marker antigen, is transcribed in a head-to-head fashion with BRCAl, with the 5' most exons of each gene located at a distance of just 295 bp.
  • the BRCAl and 1A1.3B promoter regions are highly similar but represent distinct copies of a genomic duplication.
  • One copy includes the head-to- head arrangement of the 1A1.3B gene and a putative gene with 5' sequences similar to BRCAl, referred to here as LBRCA1 (for Like BRCAl).
  • the second promoter region has a head-to-head arrangement of BRCAl with a putative gene L1A1.3B (for Like 1A1.3B) that has a 5' structure similar to 1A1.3B.
  • the data presented here demonstrate the existence of a direct genomic duplication that includes the BRCAl and 1A1.3B promoters as distinct elements.
  • the alternative forms of the duplication do not represent polymorphic variation because PCR reactions with primers specific for each distinct segment showed products of the correct size with all genomic samples (N > 90) and sequencing of such products showed the expected single pattern (data not shown). This finding has a wide variety of implications in part because it significantly revises a generally accepted (Szabo and King, 1995) and frequently cited aspect of BRCAl gene structure.
  • RNA hybridization results obtained to date with genomic and cDNA probes for 1A1.3B and BRCAl require some review. In some circumstances, evaluation of specific expression of these loci may depend on RT-PCR analysis with specific primers or the identification and verification of specific hybridization probes. More complete genomic structural characterization and transcription analysis are needed to determine the expression pattern and nature of any gene products from these loci.
  • genomic or RT- PCR primer pairs thought to be specific for BRCAl also amplify an identical sequence from LBRCA1.
  • the overall sequence similarity of ⁇ 94%, observed in the promoter regions of the two genes suggests that such confusion is not likely if this degree of similarity is representative of the entire duplication.
  • knowledge of the sequences of the two similar regions will be useful in the design of PCR primers needed for amplification of products specific for each region of the duplication.
  • L1A1.3B and not 1A1.3B is located head-to-head with BRCAl may imply a coordinate regulation and that the putative L1A1.3B gene/transcript shares a greater functional interaction or a greater developmental and tissue-specific coordination of expression with BRCAl than does 1A1.3B. Therefore, mutations in L1A1.3B could account for some instances of familial breast-ovarian cancer genetically linked to the BRCAl locus, but without any known mutation yet identified in the BRCAl gene.
  • a second gene involved in both sporadic and familial ovarian cancers that is distal to BRCAl has been inferred by loss of heterozygosity (LOH) studies (Godwin et al., 1994).
  • duplication containing all or part of BRCAl and 1A1.3B suggests that recombination events or other homology-mediated genetic rearrangements, occurring somatically or as heritable changes, could result in altered expression or inactivation of genes located within or close to the duplicated segment.
  • examples of such mechanisms include unequal exchanges resulting in the formation of chimeric genes with inappropriate expression or function (Lifton et al., 1992) and deletion or gene conversion events between highly similar gene sequences that result in non-functional arrangements (White et al., 1988).
  • the alternative possibilities of duplication or deletion of one or more genes lying between sites of homology-mediated unequal exchange may also be involved in disease etiology.
  • PMP22 gene located in proximal 17p between two homologous 24 kb elements that are separated by 1.5 Mb (Kiyosawa and Chance, 1996). Unequal exchange between these elements can cause a duplication of PMP22, resulting in Charcot-Marie-Tooth disease Type I (Pentao et al., 1992) or a deletion that causes hereditary neuropathy with liability to pressure palsies (Chance et al., 1993).
  • Genomic library screening The LANL1701 flow-sorted chromosome 17 library (Longmire et al., 1993) was provided by L. Deaven at the Los Alamos National Laboratory.
  • the vector, lambda CH40 grows in recA " bacteria, and the library has been propagated on the K802 recA " host to significantly reduce the possibility of intra- or inter-clone recombination events that might result in artifactual fusions.
  • PCR of DNA from library subpools was used to verify the presence of appropriate clones, followed by plaque hybridization. Standard methods were used for phage clone growth, DNA extraction, restriction digestion and construction of pUC8 plasmid derivatives containing each of the EcoRI fragments of each CH40 clone for further hybridization analysis and DNA sequencingr
  • Plasmid and phage clone DNAs were denatured with NaOH and applied to charged nylon filters (AMF CUNO), followed by air-drying, UV crosslinking and filter pre-washing in 0.5% SDS, 0.1 x SSC at 65°C.
  • Oligonucleotide probes were 32 P-labeled with T4 kinase and hybridized to replicate filters in 6 x SSC, 5 mM EDTA pH 8.0, 0.25% non-fat dry milk for at least two hours at 37°C, followed by three successive 5 minute washes in pre-warmed 5 x SSC, 0.1% SDS at a temperature 10°C lower than the oligonucleotide T m , as calculated by the PRIMER program. Filters were blotted dry and exposed to X-ray film for 1 to 16 days with an intensifier screen. The sequences of the oligonucleotides used are as shown in Table 1.
  • PCR primer pairs For PCR primer pairs, the thermal cycling conditions, and the specificity of the product (BRCAl or 1A1.3B) are as shown.
  • Taq polymerase and standard reaction buffer were from Promega and cycling was performed in a Perkin-Elmer 480 or Techne PHC-3 thermal cycler.
  • PCR primers 120.3 and 120.2 were designed for amplification of the BRCAl promoter region from information presented by Brown et al. (1994). This published sequence is apparently an improper fusion of lA1.3B-specific sequences with BRCAl -specific sequences, probably due to the very close similarity between the two promoter regions that caused a clone rearrangement or a false contig assignment.
  • This initial primer pair included one (120.3) that corresponds to a region of near-identity in the 1A1.3B gene with its cognate and a second (120.2) that is BRCAl -specific.
  • RE-SSCP analysis of the 1300 bp PCR product revealed two polymorphic sites.
  • PI clone 746B4 contains a segment spanning BRCAl exon 11 and the BRCAl promoter, but not including the 1A1.3B promoter, while YAC clone 173B7 includes the 1A1.3B promoter and 1A1.3B exons 12 and 19, but not the BRCAl promoter.
  • genomic clones containing the BRCAl or 1A1.3B promoters and adjacent sequences were isolated from a rearrangement-resistant lambda library. PCR analysis of the complete library DNA indicated the presence of both promoter segments. All isolated clones hybridized strongly to the 225.1 plus 225.4 PCR product used as probe, but revealed one of two distinct EcoRI restriction patterns. Clone 10A and 5 similar isolates contained two EcoRI fragments of 7.0 and 9.2 kb. Clone 16C contained EcoRI fragments of 7.1, 2.7, 2.5, 1.5 and 0.35 kb.
  • Plasmid DNAs containing individual EcoRI fragments of 10A and 16C were probed with oligonucleotides 42.2, 42.3, 225.4, 1007.5 and 214.2 (Figure 2, Table 1) to establish the fragment maps shown in Figure 1A.
  • the oligonucleotide hybridization analysis also confirmed that clone 16C includes the 5' portion of BRCAl.
  • Oligonucleotide 1007.5, corresponding to well-established BRCAl 5' cDNA sequence ( Figure 2, Table 1), showed strong hybridization to the 16C 7.1 kb EcoRI fragment but weak hybridization to the 10A 9.2 kb EcoRI fragment, detectable only with long autoradiographic exposure.
  • Figure 3 shows a dot plot of the U37574 sequence vs. a corresponding segment of clone 10A. This region includes 1A1.3B exons la and lb and BRCAl exons la, lb and 2 and their cognates. The strong diagonal elements of the plot indicate a high degree of sequence similarity across this entire length. There are three significant gaps in this similarity. Gapl ( Figure 3) is due to a 340 bp insertion in LBRCAl just at the beginning of the sequence that corresponds to BRCAl exon la ( Figure 2). It is unclear whether this insertion may be considered part of LBRCAl exon la. It does not include homology to any highly repeated human sequence.
  • Gap2 is due to an additional 61 bp of sequence within the segment of LBRCAl that corresponds to BRCAl exon lb. As indicated in Figures 2 and 3, nearly all of the LBRCAl "exon lb-like" sequence and a significant part of the BRCAl exon lb sequence correspond to a region of homology with the Alu repeat element. The additional 61 bases that are present in the LBRCAl gene represent that part of the Alu element that is missing from BRCAl exon lb. This difference strongly suggests that the Alu element at this position existed prior to the duplication event and that part of this Alu was lost in the further evolution of BRCAl exon lb.
  • exon lb is derived from an Alu element is an example of a phenomenon already described for a variety of other known genes (Makalowski et al., 1994; Baban et al., 1996). Since the Alu element is found only in primates, the proposed duplication almost certainly occurred after the genomic dispersion of this element in the primate genome. The function of exon lb in BRCAl is also very likely to be unique to primates.
  • Gap3 ( Figure 3) corresponds to a region upstream of BRCAl exon 2.
  • LBRCAl includes a complete Alu element in opposite orientation to the exon lb Alu element. This Alu is missing from the BRCAl gene. However, there are about 60 non-contiguous basepairs in BRCAl at this position that are not present in LBRCAl.
  • a fourth notable feature in Figure 3, the outlying diagonal element, is due to the presence of an additional Alu repeat, downstream of BRCAl exon 2, and in the same orientation as the exon lb element. The known sequence of the LBRCAl segment ends within this repeat.
  • 1A1.3B/LBRCA1 contains one gene with well-established transcriptional activity shows that both of the newly identified promoters are active.
  • the possibility of functional transcription of the LBRCAl and L1A1.3B genes is supported by the overall structure and sequence similarity of the promoter complex regions ( Figures 2 and 3) as well as the conservation of splicing sequences for the presumptive exons of both LBRCAl and L1A1.3B.
  • the BRCAl start site and coding frame in exon 2 are not conserved in LBRCAl, however there is a potential ATG start site close to the end of the sequence that is similar to exon 2 ( Figure 2).
  • Corresponding segments of BRCAl exon lb and LBRCAl include a perfect match to this consensus, with the expected orientation relative to the Alu. Flanking sequences distinguish both of these elements from that reported by Norris et al. (1995), showing that the 5' BRCAl region contains at least two EREs. No other consensus EREs are present in the segments shown in Figure 2.
  • IDS iduronate-2-sulphatase
  • the region containing the BRCAl and 1A1.3B genes is partially duplicated in the human genome and this duplication enhances the chance that chromosomal rearrangement will occur via unequal crossing over between the homologous elements of the duplicated structures. This could easily result in inactivation of these genes or to other pathological gene arrangements. Determining the presence of such a mutation can be more difficult than finding a point mutation and may be missed. If screening for mutations is limited to sequencing the complete coding regions of these genes, a recombination that occurred within an intron will likely not be seen. This would be the result if the gene is sequenced by first amplifying the gene via PCR using sets of primers that amplify only the exons.
  • results of such screening could well show that all of the exons are present within the genome and may find no mutations such as point mutations, insertions, deletions, etc. Nevertheless, if a recombination has occurred within the gene resulting in an unequal crossing over, at least one of the two chromosomes will in fact not have an intact gene and the gene on that chromosome will be inactive. Unequal crossing over may occur within somatic tissue or may occur in the germline. If such occurs within a cell in somatic tissue then that cell may be the start of a tumor. If the rearrangement occurs within the germline then one of the recombined chromosomes may be passed on to progeny.
  • One- method of tracking chromosomal rearrangements is to look at polymorphisms that occur within the genes.
  • BRCAl Two new polymorphisms are disclosed here. These two polymorphisms occur in the BRCAl promoter region (nucleotides 612 and 980-982 of U37574 (SEQ ID NO:l)). Nucleotide 612 may be either C or T and nucleotides 980-982 may be either AAC (as shown in U37574) or AACAAC. These two polymorphisms show a high level of linkage disequilibrium.
  • a chromosome will have either C/AAC or it will have T/ AACAAC.
  • the C/AAC haplotype has a frequency of 0.65.
  • polymorphisms may be used to track recombination within somatic tissue. If both chromosomes have the same "genotype", i.e., both are C/AAC or both are T/ AACAAC then it will be uninformative to use these polymorphisms to study recombination within BRCAl. If the person is heterozygous for these polymo ⁇ hisms, i.e., one chromosome contains C/AAC and the other chromosome contains T/ AACAAC, then use of these polymorphisms will be perfectly informative in assaying for recombination within BRCAl. To perform such an assay, germline tissue is assayed for the presence of these two polymo ⁇ hisms.
  • Somatic tissue is also analyzed. If the somatic tissue shows only one of the two genotypes then clearly the chromosome carrying the other genotype has been deleted for the region containing at least that portion of BRCAl containing the polymo ⁇ hic site. Such a result would indicate a high probability that the suspect tissue is indeed cancerous. This would be strengthened by the knowledge that the person contains a mutation known to be associated with breast and ovarian cancer. This test confirms the loss of heterozygosity which may lead to cancer when the wild-type gene is lost.
  • Such an assay is not limited to the two polymo ⁇ hisms noted above but may be used with other polymo ⁇ hisms within the BRCAl/LBRCAl region.
  • polymo ⁇ hisms have been published for BRCAl. Two new polymo ⁇ hisms within LBRCAl have been discovered and are presented here. One is at base 1723 of SEQ ID NO:2 and the other at base 2182 of SEQ ID NO:2. The base at both of these positions may be either G or A.
  • the above method is suitable for tracking recombination within somatic tissue but not in the germ line. If a person has inherited one wild-type gene and one gene with a deletion of the chromosomal region between LBRCAl and BRCAl, the person will be hemizygous for the noted polymo ⁇ hisms if the recombination has deleted them from one chromosome. This hemizygous person will appear homozygous for either the C/AAC or the T/ AACAAC polymo ⁇ hism if those are the polymo ⁇ hisms being examined.
  • a person may have three copies of the gene region containing the polymo ⁇ hisms. Such a person could be either homozygous or heterozygous for the polymo ⁇ hisms. Regardless of whether a person with a germline rearrangement has one copy or three copies of the gene region, if the recombination occurred within introns, simply sequencing exons will not discover this rearrangement.
  • the copy number of the gene region containing the polymo ⁇ hism may be determined by methods such as quantitative PCR (see, e.g., Volkenandt et al., 1992; Filliland et al., 1990; Pastore et al., 1996) or fluorescent in situ hybridization (FISH). FISH analysis would easily discern a deletion of the region. Whereas many, possibly most, genes in the human genome are not duplicated in part or whole and genetic recombination within the gene would be expected to be quite rare, BRCAl and its contiguous gene L1A1.3B are partially duplicated (as LBRCAl and 1A1.3B) and this region is therefore much more likely to undergo unequal crossing over leading to gene deletion or duplication. Analysis of such is therefore more important with BRCAl than it will be with genes which are not duplicated. The presence of the Alu repeat within the BRCA1-1A1.3B genes makes crossing over an even more likely event than for genes without such a repeat.
  • ⁇ Analysis of the copy number of BRCAl need not be limited to use of the polymo ⁇ hic regions. Any region may be used. The importance of the analysis is that if the test results indicate the presence of only a single copy then this is equivalent to having a mutation known to be associated with breast or ovarian cancer since there is only a single (at most) wild-type copy of the gene present. If the test results indicate the presence of three copies of the gene region then again this is cause for concern because it will indicate the presence of a duplication of the gene region with the possibility that the duplication is a result of unequal crossing over which has inactivated the BRCAl gene. If so then again there would be at most one copy of wild-type BRCAl present. Clearly the knowledge of copy number of the BRCAl gene is as important as knowing the presence of point mutations.
  • NAME Saxe, Stephen A.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO ⁇
  • ANTI-SENSE NO
  • CTCTACTAAA AATACAAAAA TTAGCCGGGC GTGGTGCCGC TCCAGCTACT CAGGAGGCTG 2100
  • CTGTAATCCC AGCTACTCAG GAGGCTGAGG CAGGAGAATC ACTTGAACCA GGAGGCAGAT 3540
  • AAAAAAAAAA AAAAAAAAAN AAACATGGAT GATCGGTGTC GTTGAGAGGA TAGGTATTTG 3660
  • MOLECULE TYPE DNA (genomic)
  • GTGACTGCGC GTCGTGAGCT CGCTGAGACG TTCTGGACGG GGGACAGGCC GTGGGGTTTC 1740
  • GAATTTCCGA AGCTAGGCAG ATGGGTATTC TTATGCGAGG GGCGGGGGCG GAACCTGAGA 1920
  • ACATGTCTAA TAAGATTAGG CTATTGTAAT TGCTGATTTT CTTAACTGAA GAACTTTAAA 3540
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GTAATTGTAA GCATCCTGAA ATAAAAAAGC AAGAATATGA AGAAGTAGTT CAGACTGTTA 3480
  • CTACTAGGCA TAGCACCGTT GCTACCGAGT GTCTGTCTAA GAACACAGAG GAGAATTTAT 3900

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Abstract

Les résultats d'expériences visant à détecter des polymorphismes et des mutations dans la région promotrice du gène BRCA1 ainsi que des comparaisons effectuées entre deux séquences d'ADN publiées indiquent que deux copies similaires mais distinctes de cette région existent dans le génome humain. Des amorces de PCR spécifiques pour l'amplification de chacune des deux régions promotrices ont été isolées dans des banques résistant à la réorganisation. L'analyse des séquences des clones et des produits de PCR spécifiques révèle deux réorganisations génomiques similaires de gènes tête-à-tête. Le gène BRCA1 est apposé juste à côté d'une structure de gène qui est similaire mais non identique au gène 1A1.3B et ce dernier est apposé à une structure de gène qui est fortement similaire à BRCA1 mais présente également des différences significatives. Les caractéristiques des régions promotrices de BRCA1 et 1A1.3B sont présentées dans la figure. L'analyse STS des clones YAC et P1 situés au voisinage de BRCA1 indique que ces régions promotrices similaires sont des éléments d'une duplication directe. On a émis de nouvelles hypothèses concernant des mécanismes génétiques qui peuvent être impliqués dans l'étiologie des cancers du sein et des ovaires, ces hypothèses étant fondées sur l'identification de cette structure génétique dupliquée sur le chromosome 17q. Cette invention concerne également des polymorphismes dans les gènes dupliqués, ces polymorphismes étant utiles pour la recherche de la réorganisation chromosomique de ces gènes.
PCT/US1997/021358 1996-11-26 1997-11-25 Les promoteurs des genes brca1 et 1a1.3b sont des elements paralleles d'une duplication genomique au niveau de 17q21 WO1998023779A1 (fr)

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US3110296P 1996-11-26 1996-11-26
US60/031,102 1996-11-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025190A1 (fr) * 2001-09-14 2003-03-27 The New Industry Research Organization Agent promoteur de tumeur specifique et utilisation associee

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK ON STN, Accession No. U37574, XU et al, 1996. *
GENOMICS, December 1996, Vol. 38, BARKER et al., "The BRCA1 and 1A1.3B Promoters are Parallel Elements of a Genomic Duplication at 17q21", pages 215-222. *
HUMAN MOLECULAR GENETICS, April 1994, Vol. 3, No. 4, CAMPBELL et al., "A Novel Gene Encoding a B-Box Protein within the BRCA1 Region at 17q21.1", pages 589-594. *
ONCOGENE, June 1996, Vol. 12, No. 12, BROWN et al., "The 5' End of the BRCA1 Gene Lies within a Duplicated Region of Human Chromosome 17q21", pages 2507-2513. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025190A1 (fr) * 2001-09-14 2003-03-27 The New Industry Research Organization Agent promoteur de tumeur specifique et utilisation associee
US7321030B2 (en) 2001-09-14 2008-01-22 The New Industry Research Organization Tumor-specific promotor and use thereof

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