EA011608B1 - Polymorphism in the human nbs1 gene useful in diagnostic of inherited predisposition to cancer - Google Patents

Abstract

The present invention relates to methods and kits for determining a predisposition and surveillance protocols for developing cancer, e.g., prostate and/or breast cancer due to germline mutation of the NBS 1 gene.

Description

The present invention relates generally to means and methods for diagnosing and treating a hereditary predisposition to cancer, in particular to prostate cancer and / or lobular invasive breast cancer, as well as the use of an inherited change in the ген 81 gene for diagnosing such a predisposition. In particular, the objects of the invention allow to synthesize DNA fragments and identify genomic anomalies associated with an increased susceptibility to these types of cancer.

In particular, the present invention relates to polynucleotides of molecular variants of the gene ΝΒ81. which are associated with hereditary predisposition to prostate cancer and / or lobular invasive breast cancer, as well as to vectors containing such variants (polynucleotides). In addition, the present invention relates to host cells containing such variants or vectors, and their use for the production of variants of protein ΝΒ81. In addition, the present invention relates to variants of protein ΝΒ81 and to antibodies that specifically recognize such a protein. The present invention also relates to transgenic non-human animals containing the above-described variants or vectors. In addition, the present invention relates to methods for finding and producing drugs for the treatment of cancers associated with dysfunction of the gene ΝΒ81. According to the present invention, furthermore, pharmaceutical and diagnostic compositions are proposed containing the above-described DNA variants, vectors, proteins, antibodies and drugs that can be obtained by the aforementioned methods. These compositions, in particular, are useful for the diagnosis and treatment of various diseases, in particular cancer, with drugs that are substrates, inhibitors or modulators of the gene ΝΒ81 or its product.

Some documents are cited throughout the text of this description. Each of the documents referred to herein (including any manufacturers specifications, instructions, etc.) is included in this specification by reference; however, it is not allowed that any mentioned document is in fact a prior art in relation to the present invention.

Prior art

The biochemical pathway of signaling DNA damage plays a critical role in maintaining the integrity of the genome in response to DNA damage and is involved in the pathogenesis of cancer.

Individuals with rare hereditary recessive clinical syndromes, such as the Nijmegen syndrome (ΝΒ8. Yazhedeei Lehkadekiytote), Bloom’s syndrome, Fanconi anemia, and ataxia tangiectasia (which are characterized by spontaneous chromosomal instability, immune deficiency, and yasi necrosis). DNA damage signal (1, 2). The Nijmegen syndrome is localized on chromosome 8c. | 21 (3, see also И86458534). The product of the ΝΒ81 gene (nibrin, also called p95) is a component of the 11ΜΒΕ11 / 11ΒΑΟ50 / нукле81 nuclease complex (4). This complex is part of the EC.CA1-associated genome control complex (ΒΑ8Ο, ΒΚ.СΑ1-а88ос ^ аΐе ά ά ш ш ап ап), which is responsible for the repair of DNA damage (2). The deletion of five pairs of nucleotides in exon 6 ΝΒ81 (657ll15) is present in the majority of patients with ΝΒ8 from Eastern Europe (5). It should be emphasized that these patients are homozygous carriers of the founder Nümegen syndrome mutation (allele 657e15). Heterozygous mutation carriers ΝΒ81 are common in Eastern Europe (0.6% of the total population).

Despite the long-standing efforts of researchers to develop a method for diagnosing an increased susceptibility to cancer of different localization, the need for such diagnostic methods still remains.

Accordingly, the means and methods for diagnosing and, possibly, treating an increased hereditary susceptibility to cancer are still highly desirable.

Thus, the technical task of the present invention is to meet the needs described above.

This technical problem is solved by the proposal of the objects described in the claims.

Summary of the Invention

The present invention is based on the discovery of a new, still unknown correlation between one form of the gene ΝΒ81 and an increased hereditary predisposition to certain types of cancer.

Unexpectedly, according to the invention defined in the claims, it has been found that the gene 81 may play a role in the pathogenesis of prostate cancer (9) or invasive carcinoma of the mammary gland of a lobular subtype. Unexpectedly, it was found that heterozygous carriers of the founding mutation of the Nijmegen syndrome (allele 657e15) may be present at an increased risk of cancer, especially prostate cancer and breast cancer.

Based on the discovery of this phenomenon, diagnostic tests and reagents for such tests for specific detection and genotyping of alleles ΝΒ81 in humans have been developed. Definition of allelic

- 1,011,608 of the state of the gene ΝΒ81 in humans using such tests may be useful for the prevention or treatment of various diseases, especially cancer, with drugs that are substrates, inhibitors or modulators of the gene product ΝΒ81.

According to the first embodiment, the invention provides polynucleotides of molecular variants of the gene ΝΒ81, correlated with a hereditary predisposition to cancer, in particular to prostate cancer or lobular invasive breast cancer, and related embodiments of the invention, such as vectors, host cells, variants of proteins ΝΒ81 and methods for their preparation.

According to another embodiment of the invention, methods are provided for identifying and obtaining drug candidates and modulators, such as inhibitors No. 81, for treating or preventing cancer, as well as methods for diagnosing the condition of such disorders / predisposition.

According to a further embodiment of the invention, pharmaceutical and diagnostic compositions are proposed, containing the polynucleotides described above, containing their vectors, proteins, antibodies to them, as well as drugs and inhibitors that can be obtained by the aforementioned method.

Pharmaceutical and diagnostic compositions, methods and uses of the invention are useful for diagnosing and treating / preventing a hereditary susceptibility to cancer.

Detailed Description of the Invention

Detection of variations in the ΝΒ81 gene, correlating with hereditary susceptibility to cancer, in particular to prostate cancer or lobular invasive breast cancer, and diagnostic tests for recognizing various ΝΒ81 alleles in human individuals provide an effective tool for improving therapy and / or preventing cancer.

Accordingly, the invention relates to a polynucleotide associated with an increased hereditary predisposition to cancer, in particular to prostate cancer or invasive breast cancer of a lobular subtype, wherein this polynucleotide is selected from the group consisting of: (a) a polynucleotide having a nucleic acid sequence of any of 8EO GO ΝΟ :, either (b) a polynucleotide encoding a polypeptide, having the amino acid sequence of any of 8EO GO ΝΟ :, or (c) a polynucleotide encoding a molecular variant of the polypeptide ΝΒ81, where the specified polynucleotide contains a nucleotide substitution, nucleotide deletion, additional nucleotide or additional nucleotide and nucleotide substitution in the position corresponding to the position 18155, 18156, 18157, 18158 or 18159 of the gene ΝΒ81 (Oepiac, registration number: АВ013139); (d) a polynucleotide encoding a molecular variant of the ΝΒ81 polypeptide, where the specified polynucleotide contains a deletion of the nucleotide in the position corresponding to the position 18155, 18156, 18157, 18158 or 18159 of the gene ΝΒ81 (Oepiac, registration number: АВ013139); (e) a polynucleotide encoding the molecular variant of peptide ΝΒ81, where the specified polypeptide contains a deletion of the amino acid at the position from 234 to 754 of the polypeptide ΝΒ81 (8ЕО ru): 219, O1p on Leis in position 220, 11e on O1p in position 221, P1e on Agd in position 222, Luc on O1i in position 223, O1u on Akp in position 224, Luck on 11e in position 225, T1n on Tug in position 226 , P1e on 11e in position 227, 11e on P1e in position 228, P1e on O1i and in position 229, Lei on Suk in position 230, Acn on O1p in position 231, A1a on T1g in position 232, b on A1a in position 233 of the polypeptide ΝΒ81 (8ЕО ГО ΝΟ: 2) and (е) of the polynucleotide encoding the molecular variant of the polypeptide ΝΒ81, having the amino acid sequence 8ЕО ГО ΝΟ: 4.

In the context of the present invention, the term "molecular variant" of a gene or protein ΝΒ81, as used here, means that the indicated gene or protein ΝΒ81 differs from the wild-type gene белка81 (genomic sequences of the gene ΝΒ81 are described, for example, under registration number AB013139) of a nucleotide (s) replacement (s), insertion (s) and / or deletion (s). Preferably, the result of this nucleotide substitution is the presence of a different amino acid in the amino acid sequence of protein ΝΒ81, the predicted result of which is a dysfunction of this protein.

The term "appropriate", as used here, means that the position is determined not only by the number of preceding nucleotides and amino acids, respectively. The position of a nucleotide or amino acid, which may be missing, be replaced or contain one or more additional nucleotides, in accordance with the present invention may be different due to deletions or additional nucleotides or amino acids elsewhere in the gene or polypeptide. Therefore, under the "appropriate position" according to the invention should be understood that the nucleotides or amino acids may differ in the specified number, but they may have the same neighboring nucleotides or amino acids. These nucleotides or amino acids, which can be replaced, absent or contain additional nucleotides or amino acids, are also included in the term "corresponding position". Such nucleotides or amino acids can, for example, together with their neighbors form sequences that may be involved in the regulation of gene expression, the stability of the corresponding RNA or RNA splicing, and also encode functional domains or motifs of the protein according to the invention.

In accordance with the present invention, the genetic variant of the gene ΝΒ81 was investigated by analyzing the sequence of the relevant regions of the human ΝΒ81 gene. A well-known fact is

- 2,011,608 that the genomic DNA of individuals who carry the individual genetic structure of all genes, including ΝΒ8, can be easily isolated from individual blood samples. These individual DNA samples are then used to analyze the sequence of the ΝΒ81 gene alleles that are present in the individual who provided the blood sample. Sequence analysis can be performed using PCR amplification of relevant regions of the gene ΝΒ81, subsequent purification of PCR products with subsequent automatic sequencing using traditional methods (cyclic sequencing with a dye on the AB1 terminator).

One of the important parameters that should be considered when trying to determine the individual genotype ΝΒ81 (and, possibly, identify new variants of the gene ΝΒ81) is the fact that each person carries (with very few abnormal exceptions) two copies of the gene ΝΒ81 two alleles 81 (diploidy ), one inherited from the mother, and the other from the father. In this regard, some individuals carry a mutation in only one copy (allele) of a gene (heterozygous mutation carriers). These patients are healthy and do not have an abnormal phenotype. Rare individuals, on the contrary, have mutations in both alleles (homozygous carriers of mutation ΝΒ81). They are affected by syndrome 8, which is characterized by phenotypic abnormalities, immunodeficiency, as well as a predisposition to cancer, especially malignant tumors of the lymphatic system. It should be noted that the present invention focuses on the detection of susceptibility to cancers among heterozygous carriers of the ΝΒ81 mutation. An example of a mutation in the ΝΒ81 gene identified as correlating with a hereditary predisposition to cancer, in particular prostate cancer or lobular invasive breast cancer in accordance with the present invention, is 657b15. Mutation assays are performed using standard techniques and described in detail in the examples. Genetic testing of population variability in relation to genetic markers of hereditary susceptibility to cancer is proposed as a tool useful for identifying and selecting patients who may have such a predisposition. This identification / selection can be based on the molecular diagnosis of genetic polymorphism by genotyping DNA isolated, for example, from a patient's leukocytes, and determining the characteristics of a possible predisposition. For healthcare founders, such as US healthcare organizations and government health services in many European countries, this pharmacogenetic approach can be both a way to improve healthcare and a way to reduce costs, because cancer therapy is expensive.

The result of mutations in variants of the ΝΒ81 genes is sometimes (are) amino acid (deletion) (s), insertion (s) and, in particular, replacement (s) either individually or in combination. Of course, it is also possible to create such mutations by genetic engineering in wild-type genes or in other mutant forms. Methods for introducing such modifications into the DNA sequence of the gene ΝΒ81 are well known to specialists in this field; see, for example, Zatzgokok, Methodology Group: AbotaFgu Mapia1, Co1b Zrtshd Nagyog Baotataot (1989) ..

In a preferred embodiment of the invention, the polynucleotide described above encodes a variant of protein ΝΒ81 or its fragment, for example, containing one or more epitopes of the amino acid sequence encoded by 8EO GO ΝΟ: 4.

To study the nature of changes in the amino acid sequence of protein ΝΒ81, you can use computer programs, such as BKA.8M0B, which can be obtained online. In addition, models of assembly and computer-aided reconstruction of structural motifs can be carried out using other suitable computer programs (01кхе \\\\\ ct Proc 25 (1996) @ 286-299; NoGGshap. Wipe !. App1. 679). Computers can be used for conformational and energy analysis of detailed protein models (Mopda, 1. Mo1. Bu1. 247 (1995), 995-1012; KepoiG, Αάν. Exp. Meb. Bu1. 376 (1995), 37-45). These assays can be used to identify the effect of a particular mutation on the binding and / or interaction with other members of the NMKE11 / UCAO50 / MB81 nuclease complex.

Typically, said amino acid deletion, insertion, or substitution in the amino acid sequence of a protein encoded by a polynucleotide of the invention is the result of one or more nucleotide substitutions, insertions or deletions, or any combinations thereof. Preferably, the result of the specified nucleotide deletions, insertions or substitutions is the deletion of the amino acid at positions from 234 to 754 of the ΝΒ81 polypeptide (8ЕО ГО Ν0: 2) and, possibly, at least one replacement of the amino acid selected among the replacement of Beech for Acn at position 219, С1п by Lei in position 220, 11 on С1п in position 221, Р1е on Agd in position 222, Beech on С1и in position 223, С1у on Akp in position 224, Beech on 11е in position 225, Т1г on Tug in position 226, Р1е on 11е in position 227, 11e on Р1е in position 228, Р1е on С1и in position 229, Bei on Suk in position 230, AcP on С1п in position 231, А1а on Т1г in position 232, Beech on A1a at position 233 of the polypeptide ΝΒ81 (8ЕО ΙΌ Ν0: 2). A polynucleotide of the invention may additionally contain at least one nucleotide and possibly amino acid deletion, insertion and / or substitution, such as insertion and substitution deletions, described and known from the prior art than the above. This embodiment of the present invention enables the study of the synergistic effects of the mutations of the gene ΝΒ81 in relation to the hereditary susceptibility to cancer in patients carrying

- 3,011,608 such mutant forms of this gene or similar mutant forms that can be mimicked by the proteins described above. It is expected that the analysis of these synergistic effects will provide a deeper understanding of the mechanism of the occurrence of hereditary susceptibility to cancer. Based on this deeper understanding, the development of diagnostic and pharmaceutical compositions related to cancer will greatly benefit.

Even more noteworthy is that when ΝΒδ-dependent types of cancer there is no functional copy of the gene (see Example 3: LON). Thus, only the mutant copy remains in the cancer cell. Therefore, for example, inhibition of the mutant allele can lead to a complete loss of protein ΝΒ81 from the cell. This should cause the death of the cancer cell. This is one of the possible therapeutic goals of an embodiment of the present invention.

Thus, in a preferred embodiment, the present invention relates to polynucleotides of molecular variants of the gene ΝΒ81, where the result of nucleotide deletion, insertion and / or replacement is altered expression of the variant gene ΝΒ81 compared with the corresponding wild-type gene.

A polynucleotide of the invention may be, for example, DNA, cDNA, genomic DNA, RNA, or synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecule containing either of these polynucleotides, either alone or in combination. Preferably, said polynucleotide is part of a vector, in particular, plasmids commonly used in genetic engineering, cosmid, viruses, and bacteriophages, which contain the polynucleotide of the invention. Such vectors may contain additional genes, for example marker genes, which allow the selection of said vector in a suitable host cell and under suitable conditions.

In a further preferred embodiment of the vector of the invention, the polynucleotide of the invention is functionally linked to expression regulatory sequences that allow expression in prokaryotic or eukaryotic cells. Expression of said polynucleotide involves the transcription of this polynucleotide, preferably into the translated mRNA.

Regulatory elements that provide expression in eukaryotic cells, preferably in mammalian cells, are well known to those skilled in the art. They usually contain regulatory sequences that provide transcription initiation and, possibly, poly-A signals that provide for transcription termination and transcript stabilization. Additional regulatory elements may include both transcriptional and translational enhancers. Possible regulatory elements that provide expression in prokaryotic host cells include, for example, the 1ac, τρ or 1ac promoter in E. so11, and examples of the regulatory elements that provide expression in eukaryotic host cells are the AOX1 promoter or 6AE1 in yeast or SMU- , 8U40-, B8U-promoter (Rous sarcoma virus), smu-enhancer, 840-enhancer or globin intron in mammalian cells and other animals. In addition to the elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals, such as the poly-A site 8U40 or the poly-A site 1k. "Downstream" from polynucleotide. Expression vectors suitable in this context are known in the art, they are, for example, the Okauata Shegd pcEU1 (Ryagtas1a) cDNA expression vector, pRIM8, pBc / SMU, ρc ^ NA1, ρc ^ NAA3 (1yyyy), p8PPT1 (ΟΙΒΟΟ ΒΒΕ). Preferably, said vector is an expression vector and / or a gene transfer vector or targeting vector. Expression vectors derived from viruses, such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, can be used to deliver polynucleotides or a vector of the invention to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Scratum, Methodology Group: A Logogu Mapia1, Collision Scratch Nagyog Logur (1989) Ν.Υ., and Auschie1, Step! Group of Companies of the Group of Companies, Group of Directors of the A55aaAle5 App \ UPEU 1n1s8s1eps, .Υ. (1994). Alternatively, the polynucleotides and vectors of the invention may be placed in liposomes for delivery to target cells.

The present invention further relates to host cells transformed with a polynucleotide or vector of the invention. Such a host cell may be a prokaryotic or eukaryotic cell, see above. A polynucleotide or vector of the invention located in such a host cell can either be integrated into the genome of this host cell or it can be maintained extrachromosomally. In this regard, it should also be understood that the recombinant DNA molecule of the invention can be used to "direct the gene" and / or "replace the gene", to reconstruct the mutant gene, or to create a mutant gene through homologous recombination; see, for example, Moiehs, Prgos. № and. Asab. 8cr I8A (1990) 4712-4716; 1pepeg, Sepa Tagdebd, And RgasIsa1 Arrgoasy, Ohgog Ishuegayu Regge§8.

The host cell may be a prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred fungal cells are, for example, cells of the fungus of the genus Massiagotus 5, in particular of the species 8. segehyae. The term "prokaryotic" means including all bacteria that can be transformed or trans

- 4 011608 to be expressed by polynucleotide for expression of variant варианта81 protein or its fragment. Prokaryotic hosts can include both gram-negative and gram-positive bacteria, such as, for example, E. Soi, 8. Ινρΐιίιηιιππιιιη. 8gabba tagseksepk and VasShik KiYShk. A polynucleotide encoding the mutant form of the белков81 variants of proteins can be used to transform or transfect a host using any of the techniques commonly known to those of ordinary skill in the art. Methods for producing fused, functionally related genes and their expression in bacterial or animal cells are well known in the art (8Hgoc, see above). The genetic constructs and methods described here can be used to effect the expression of protein variants ΝΒ81, for example, in prokaryotic hosts. In general, expression vectors containing promoter sequences that promote efficient transcription of the polynucleotide of the insert are used in conjunction with the host. The expression vector usually contains an origin of replication, a promoter and a terminator, as well as specific genes that are capable of providing phenotypic selection of transformed cells. Transformed prokaryotic hosts can be grown in fermenters and cultured according to methods known in the art to achieve optimal cell growth. Then the proteins according to the invention can be isolated from the growth medium, from cell lysates or cell membrane fractions. Isolation and purification of the polypeptides of the invention expressed in microbes or in other organisms can be performed by any conventional means, such as, for example, separation by preparative chromatography, which, for example, involves the use of monoclonal or polyclonal antibodies.

Thus, in a further embodiment, the invention relates to a method for producing variants of proteins ΝΒ81 and their fragments, in which a host cell is cultured, as defined above, under conditions enabling the expression of the protein, and the produced protein or its fragment from the culture is isolated.

In another embodiment, the invention relates to a method for producing cells capable of expressing a variant of the gene 81, including the creation of cells using a genetic engineering method, including a polynucleotide or with a vector of the invention. The cells obtained by the method of the invention can be used, for example, to test drugs by the methods described by I.L. 8ress, Β.Ό. So1btap, L.L. Testapb, Ce11k, and LAF tapi-1, C8H Prégc 1998. In addition, these cells can be used to study known drugs and their unknown derivatives for their ability to complement the disorder caused by mutations in the ΝΒ81 gene (for example, susceptibility to malignant tumors). For these embodiments, there is preferably no wild type allele in the host cells, preferably both alleles of the ΝΒ81 gene and / or at least one of them is mutated. Or, a strong overexpression of the mutated allele with respect to the normal allele and comparison with a recombinant cell line expressing the normal allele at a similar level can be used as a screening and analysis system. Cells that can be obtained by the method described above can also be used for screening by the methods described below.

In addition, the invention relates to the variant protein ΝΒ81 or its fragments encoded by the polypeptide according to the invention, either obtained by the methods described above, or obtained from cells constructed by the method described above.

In this context, it is also clear that variants of proteins ΝΒ81 according to the invention can be further modified by conventional methods known in this field. Using variants of proteins ΝΒ81 according to the present invention, it is also possible to determine areas relevant for their biological activity or inhibition thereof, namely, by their participation in the formation of the correct nuclease complex ΗΜΚΕ11 / ΚΚΑΌ50 / ΝΒ81.

The present invention further relates to antibodies that specifically recognize the variant protein ΝΒ81 according to the invention. Preferably, this antibody specifically recognizes an epitope containing one or more amino acid substitutions, as described above. Antibodies against variant protein белка81 according to the invention can be obtained by well known methods using the purified protein according to the invention or its (synthesized) fragment as an antigen. Monoclonal antibodies can be obtained, for example, using techniques that were first described in KoSheg apb M11klesh, No. 11ig 256 (1975) 7 495, and Sa116, Mech. Ephuto1 73 (1981) 9 3, and include the fusion of mouse myeloma cells with spleen cells isolated from immunized animals. Antibodies can be monoclonal antibodies, polyclonal antibodies or synthesized antibodies, as well as antibody fragments, such as EA, Ыν or csE fragments, etc. In addition, antibodies to the aforementioned polypeptides or fragments thereof can be obtained by methods that are described, for example, in the Pharmaceutical Program, Antibiotic, A, Laurel, Computeria, C8H Prgcc, and Coballobacter, 1988.

These antibodies can be used, for example, to immunoprecipitate and immunolocalize variants of proteins ΝΒ81 according to the invention, as well as to monitor the presence of such variants of proteins ΝΒ81, for example, in recombinant organisms, as well as to identify compounds that interact with proteins of the invention. For example, plasmon surface resonance, which is used in the B1Asoge system, can be used to enhance the efficiency of phage antibodies that bind to the epitope of the protein of the invention (Cyster, Nitap UpbObTyrBottac 7 (1996), 97-105; MacroTec, 1. 1 ttypo1. MeShop 183 (1995) I 7-13). In addition, the present invention relates to Nuk molecules.

- 5,011,608 leic acids, which are or contain a complementary strand of any of the above polynucleotides or a part thereof, thus having a difference of at least one nucleotide compared with the corresponding wild-type 81 gene nucleotide sequences, namely, having the above-described nucleotide substitutions, deletion and insertion. Such a molecule can either be deoxyribonucleic acid or ribonucleic acid. Such molecules include, for example, antisense RNA. These molecules can also be linked to sequences that, when transcribed, encode a ribozyme, thus producing a ribozyme that specifically cleaves the transcripts of the polynucleotides of the invention. In addition, the present invention relates to a vector containing a nucleic acid molecule according to the invention. Examples of such vectors are described above.

Preferably, the nucleic acid molecule present in the vector is functionally linked to regulatory elements that allow expression in prokaryotic or eukaryotic host cells; see above.

The present invention also relates to a method for creating a transgenic animal that is not a human, preferably a transgenic mouse, in which a polynucleotide or vector of the invention is introduced into a germ cell, an embryonic cell, a stem cell, or an egg cell, or a cell derived from them. An animal that is not human can be used in the method of the invention described below, and it may be a non-transgenic healthy animal or it may have a disorder, preferably a disorder caused by at least one mutation in the 81 gene. Such transgenic animals are well suited, for example, for pharmacological studies of drugs in connection with the variant forms of the above-described variants of proteins No. 81, since these proteins, or at least their functional domains, are conservative between species in higher eukaryotes, in particular in mammals. The production of transgenic embryos and their screening can be carried out, for example, as described by A. b. 1, Eb., Spsp Tagdsypd, A Rtasyis1 Arrgoasy (1993), OhRotb Ishusgayu Rgskk. Embryo DNA can be analyzed by conducting, for example, Southern blots with an appropriate probe.

The invention also relates to non-human transgenic animals, such as, for example, transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, C. clips, and fish, such as, for example, electric rays, containing a polynucleotide or vector according to the invention, or obtained as described above, preferably where the specified polynucleotide or vector is stably integrated into the genome of the specified animal, not a human being, preferably in such a way that the presence of the specified polynucleotide or vector leads to the expression of the protein ΜΌΚ embodiment of the invention. An animal may have one or more copies of this or other polynucleotides of the ΝΒ81 gene variant. This animal can have various applications, including the use as a research model of a malignant process, and, therefore, is a new and valuable animal in the development of therapies, treatment, etc. of diseases caused by a deficiency or insufficiency of the correct repair of DNA damage in the cell. Accordingly, in this case, the animal is preferably a laboratory animal, such as a mouse or a rat.

Preferably, the transgenic non-human animal of the invention further comprises at least one inactivated wild type allele of the ΝΒ81 gene. This embodiment makes it possible, for example, to investigate the interaction of various variant forms of proteins ΝΒ81. It may also be desirable to inactivate the expression or function of the ΝΒ81 gene at a certain stage of development and / or life of this transgenic animal. This can be accomplished, for example, using tissue-specific, developmental and / or cell-regulated and / or inducible promoters that direct expression, for example, antisense RNA or a ribozyme directed against the 81 gene RNA transcript; see also above. A suitable inducible system is, for example, gene expression regulated by tetracycline, as described, for example, Socxp apb Bi) agb (Proc. Na11. Asab. 8c1. 89 I8A (1992), 5547-5551 and Socxp with! A1. (Tsspbk By1ssy 12 (1994), 58-62). Similarly, expression of the variant 81 gene can be regulated by such regulatory elements.

In the presence of variants of polynucleotides ΝΒ81, as well as proteins and vectors according to the invention, it is now possible to investigate the w ί v \ ί and w ν 11 th efficiency of DNA damage repair for specific mutations in the ген 81 gene.

In addition, variants of proteins ΝΒ81 can be used to determine the pharmacological profile of anticancer drugs and to identify and obtain additional drugs that may be more effective for treating cancer, in particular, to attenuate some phenotypes caused by appropriate mutations, such as described above.

The following embodiment of the invention relates to a method for identifying and obtaining a modulator ΝΒ81 capable of modulating the activity of the molecular variant of the gene ΝΒ81 or its gene product, in which: (a) a protein according to claim 8 is brought into contact or a cell expressing the molecular variant of gene ΝΒ81 containing According to claim 1 or 2, in the presence of components capable of providing a detectable signal in response to the activity of protein ΝΒ81, with the compound to be

- 6,011,608 screening, under conditions that allow the activity of protein ΝΒ81 to be displayed; and (b) determine the presence or absence of a signal or the increase in the signal generated by the activity of protein ΝΒ81, where the presence or increase of the signal is an indicator of the intended modulator.

The term “compound” in the method of the invention includes one substance or a plurality of substances that may be the same or different.

Said compound (s) may be chemically synthesized or produced by fermentation of microorganisms, and may also be contained, for example, in samples, for example, in cell extracts, for example, of plants, animals or microorganisms. In addition, these compounds may be known in the art, but are still unknown as useful as an inhibitor, respectively. Many of the compounds can, for example, be added to the culture medium or injected by them with a cell or an animal according to the invention, which is not human.

If a sample containing (a) compound (s) is identified in the method of the invention, then it is possible to either isolate this compound from the original sample identified as containing the compound of interest, or the original sample can be further separated, for example, if it consists of many different compounds , so as to reduce the number of different substances per sample, and repeat this method with parts obtained by separating the original sample. You can then determine whether the specified sample or compound exhibits the desired properties, for example, by the methods described in this document or in the literature (8 c1 a1., Ce11k tapi-1; see above).

Depending on the complexity of the samples, the above steps can be carried out several times, preferably until the sample identified by the method of the invention contains only a limited number of substances or only one substance.

Preferably, said sample contains substances with similar chemical and / or physical properties, and most preferably, said substances are identical. The methods of the present invention can be easily implemented and planned by a person skilled in the art, for example, using other cell-based assays described in the prior art, or by using and modifying the methods described herein. In addition, the person skilled in the art can easily understand which additional compounds and / or enzymes can be used to implement the methods of the invention, for example enzymes, if necessary, which convert a specific compound into its precursor, which, in turn, is a substrate for squirrel ΝΒ81. Such an adaptation of the method according to the invention is within the competence of a person skilled in the art and can be carried out without undue experimentation.

Compounds that can be used in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, ΡΝΑ, and the like. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described, for example, in WepChesh. Drink about! Ogdashs Syetu. 8rgshdeg eShop № \ ν Wagk 1ps., 175 Iyi Auepie, № \ ν Watk, Ν.Υ. 10010 I8A, and in Ogadash Gup1yek1K, ^ Yeu, № \ ν Укк, И8А. In addition, these derivatives and analogs can be tested for their effects by methods known in the art, or as described in this document. In addition, peptidomimetics and / or computer design of derivatives and analogs of the respective drugs can be used, for example, in the methods described below. Such analogs include molecules that are based on the structures of known MBE substrates and / or inhibitors and / or modulators, see below.

Appropriate computer programs can be used to identify the interactive sites of the intended modulator / inhibitor and protein ΝΒ81 according to the invention using computer programs for searching complementary structural motifs (Eakksha, Tipster 5 (1994), 114120). Additional suitable computer systems for computer-aided design of proteins and peptides are described in the art, for example, in Weggu, Wusset. 8os Tgapk. 22 (1994) 7 1033-1036; ^ oyak, App. Ν.Υ. Asay. 8c1. 501 (1987) 2 1-13; PaBo, WusjetMgu 25 (1986), 5987. The results obtained on the basis of the computer analysis described above can be used in conjunction with the method of the invention, for example, to optimize known modulators / inhibitors. Suitable peptidomimetics and other inhibitors can also be identified by synthesizing combinatorial libraries of peptidomimetics using sequential chemical modification and testing of the resulting compounds, for example, using the methods described here. Methods for the preparation and use of combinatorial peptide mimetic libraries are described in the prior art, for example, in Oyteky, Meyloi8 and Ephthoid 267 (1996), 220-234, and Botet, Vyuotd. May Siet 4 (1996), 709. In addition, the three-dimensional and / or crystallographic structure of inhibitors and protein No. 81 according to the invention can be used for the design of peptidomimetic drugs (Voko, VyuyetMgu 35 (1996), 1293312944; Vylepbet, Vyuotd. May. Siet. 4 ( 1996), 1545-1558).

In summary, the present invention provides methods for identifying and preparing compounds that can be used to treat / prevent cancer.

In the following embodiment, the present invention relates to a method for identifying and obtaining a P81 modulator capable of modulating the activity of the molecular variant of gene BB81 or its gene product, in which: (a) the protein of claim 8 is brought into contact with the first molecule known

- 7,011,608 as being bound by protein ΝΒ81, with the formation of the first complex containing the indicated protein and the indicated first molecule, (b) bringing the said first complex into contact with the compound to be screened, and (c) measuring whether the indicated compound displaces the said first molecule specified first complex.

Preferably, in this method, said measurement step comprises measuring the formation of a second complex of said protein with said candidate modulator compound.

Preferably, said measurement step comprises measuring the amount of said first molecule that is not bound to said protein.

In addition, it is preferable that in the method according to the invention, said first molecule is labeled, for example, with a radioactive or fluorescent label.

In another embodiment, the present invention relates to a method for diagnosing an increased hereditary susceptibility to cancer, wherein: (a) determine the presence of a polynucleotide of the invention in a sample taken from a subject, and / or (b) determine the presence of a variant protein ΝΒ81, for example, with an antibody according to the invention.

In accordance with this embodiment of the present invention, a method for testing a hereditary susceptibility to cancer can be accomplished by using a polynucleotide or nucleic acid molecule according to the invention, for example, in the form of a Southern or Northern blot, or an к kyi assay. The specified nucleic acid sequence can be hybridized with the coding region of any of the genes or with a non-coding region, for example with an intron. In the case when the complementary sequence is used in the method according to the invention, said nucleic acid molecule can be reused in Northern blots.

In addition, the specified testing can be carried out in connection with the actual blocking, for example, transcription of a gene, and, therefore, expect that it has therapeutic relevance.

In addition, the primer or oligonucleotide can also be used for hybridization with one of the above-mentioned genes 81 or the corresponding mRNA. Nucleic acids used for hybridization, of course, can be conveniently labeled by incorporating or attaching, for example, a radioactive or other marker. Such markers are well known in the art.

The labeling of these nucleic acid molecules can be accomplished by conventional methods.

In addition, the presence or expression of variants of the ΝΒ81 genes can be monitored using a pair of primers that specifically hybridize with any of the corresponding nucleic acid sequences, and PCR reactions are carried out according to standard methods. Specific hybridization of the above probes or primers preferably occurs under stringent hybridization conditions. The term “stringent hybridization conditions” is well-known in the field, see, for example, Silvio S1 a1., Computer Science, A Logus Für Mapia 1 Cesopia, S8H Pragk, Cole 8rppd Nagyog, 1989; No. 1 S1Al AiNi Nypi1a1iup, A Rgasbsa ArrgoasN, Natek Api Neschpk eik. mutations of the gene ΝΒ81. Further, the present invention includes methods where such a fingerprint can be created using restriction fragment length polymorphism (BELI) of DNA or RNA obtained from a subject, perhaps DNA or RNA can be amplified before analysis by methods well known in the art. RNA fingerprints can be obtained, for example, by enzymatic hydrolysis of a sample of RNA obtained from a subject, an enzyme suitable for RNA, for example, RNase T1, RNase T2, etc., or a ribozyme and, for example, electrophoretic separation and detection of RNA fragments as described above .

Further modifications of the above embodiment of the invention can be easily undertaken by a person skilled in the art without undue experimentation based on the description of the invention; see, for example, examples. A further embodiment of the present invention relates to a method in which said determination is carried out using an antibody according to the invention or a fragment thereof. The antibody used in the method of the invention may be labeled with detectable labels, such as his-tag flags or biotin molecules.

In a preferred embodiment of the present invention, the above described methods comprise PCR, ligase chain reaction, restriction enzymatic hydrolysis, direct sequencing, a technique of nucleic acid amplification, microchips, hybridization techniques or immunoassays (8atgook e1 a1., 1 ° C. C11. S8N s1oshpd, Nag1oh apy Lane 1 ° C .11 C8H apyoShek).

In a preferred embodiment of the method of the present invention, said cancer is prostate cancer or invasive breast cancer of a lobular subtype.

In another embodiment of the method described above, the next stage comprises administering to the subject a medicament for removing or attenuating said cancer.

In a preferred embodiment of the method according to the invention, said drug is a chemotherapeutic agent, such as adriamycin, doxorubicin, paclitaxol (taxol) and other MOV-substrates, Athycag 8U. e1 a1., Appi. Wow. Ryagtaso1. Toh1so1. 39 (1999) 9 361. In another preferred embodiment of the methods described above, this method further includes an introduction to

- 8,011,608 cells: (ί) functional and expressed wild-type gene ΝΒ81 or () nucleic acid molecule or vector of the invention.

In this context, and also as used throughout the description of the invention, the term “functional” gene ΝΒ81 means a gene where the encoded protein has part or all of the primary structural conformation of the wild-type protein 81, that is, it has the biological property of participation in the nuclease complex ΗΜΚΕ11 / ΗΚΑΌ50 / ΝΒ81 with the right activity. This embodiment of the present invention is suitable for the treatment / prevention of cancer. Detection of expression of a variant of the gene позволит81 will lead to the conclusion that this expression is interconnected with a predisposition or with the maintenance of the corresponding cancer phenotype. Accordingly, this stage will be used to reduce the expression level to low levels or to stop expression. This can be done, for example, by at least partially eliminating the expression of the mutant gene by biological methods, for example, using enzymes, antisense nucleic acid molecules, intracellular antibodies or the above described inhibitors against these variant proteins ΝΒ81.

In addition, it is possible to develop pharmaceutical products that reduce the expression level of the corresponding mutant proteins and genes.

In a further embodiment, the invention relates to a method for preparing a pharmaceutical composition, comprising the steps of any of the methods described above and the step of synthesizing and / or incorporating into the preparation the compound identified in step (b) or its derivative or homolog in a pharmaceutically acceptable form. Therapeutically useful compounds identified according to the method of the invention can be incorporated into a preparation and administered to a patient, as discussed above. Applications and therapeutic dosages defined by a person skilled in the art as suitable, see below.

Further, the present invention relates to a method for preparing a pharmaceutical composition, comprising the steps of any of the above methods and the steps of incorporating a drug or prodrug into a preparation in a form suitable for therapeutic use and preventing or alleviating the disorder in a subject diagnosed by the method of the invention.

Drugs or prodrugs after their administration ίη νίνο undergo transformation during metabolic processes in order to remove them either by isolating or by turning into one or more active or inactive metabolites (Meueg, 1. PHTasocytes Βίορίιαηη. 24 (1996), 449-459) . Therefore, instead of the compound itself or the inhibitor identified and obtained by the methods of the present invention, rather, it is necessary to use the corresponding drug in the form of a prodrug that is converted to its active form in the patient's body. The precautions that can be taken to use prodrugs and drugs are described in the literature; see review in Ohata. 1. Toh1so1. 8c1. 21 (1996), 323-329. In a preferred embodiment of the method of the present invention, said drug or prodrug is a derivative of the drug as defined above.

In another embodiment, the present invention relates to an inhibitor identified or obtained by the method described above. Preferably, this inhibitor binds specifically to the variant protein ΝΒ81 of the invention. Antibodies, nucleic acid molecules and inhibitors of the present invention preferably have a specificity of at least substantially identical binding specificity of the native ligand or protein binding partner # 81 of the invention. The antibody or inhibitor may have a binding affinity for the protein ΝΒ81 according to the invention of at least 10 ⁵ M ^-1 , preferably higher than 10 ⁷ M ^-1 and most preferably up to 10 ¹⁰ M ^-1 in the case where activity ΝΒ81 must be suppressed. Therefore, in a preferred embodiment, the suppressor antibody or inhibitor of the invention has an affinity of at least about 10 ⁻⁷ M, preferably at least about 10 ⁻⁹ M, and most preferably at least about 10 ⁻¹¹ M.

In addition, the present invention relates to the use of an oligo or polynucleotide for detecting a polynucleotide of the invention and / or for genotyping the corresponding individual alleles ΝΒ81. Preferably, said oligo- or polynucleotide is a polynucleotide or nucleic acid molecule according to the invention described above.

In a particular preferred embodiment, the length of said oligonucleotide is from about 10 to 100, more preferably from 15 to 50 nucleotides, and it contains the nucleotide sequence included in one of 8EC ΙΌ ΝΟ: 1, 8EC: 3, 8EC ΝΟ: 5- 12 or a sequence complementary to any of them.

Therefore, in yet another embodiment, the present invention relates to a primer or probe consisting of an oligonucleotide as defined above. In this context, the term "consisting of" means that the nucleotide sequence described above and used for the primer or probe according to the invention does not have any additional nucleotide sequences of the gene 81, directly adjacent to its 5 'or 3' end. However, other groups, such as tags, such as biotin molecules, histidine flags, antibody fragments, colloidal gold, etc., as well as nucleotide sequences that do not correspond to gene ΝΒ81, may be present in the primer and probes of the invention. In addition, it is also possible to use specific

- 9,011,608 the nucleotide sequences described above and combine them with other nucleotide sequences derived from the gene ΝΒ81, where these additional nucleotide sequences are distributed between groups other than nucleic acids, or where the nucleic acid does not correspond to the nucleotide sequences of the gene ΝΒ81.

In addition, the present invention relates to the use of an antibody or substance capable of specific binding to the gene product of the gene ΝΒ81, for detecting a variant of the protein ΝΒ81 according to the invention, expressing the molecular variant of the gene ΝΒ81 containing the polynucleotide of the invention, and / or for distinguishing alleles ΝΒ81 containing polynucleotide according to the invention.

The present invention also relates to a composition, preferably a pharmaceutical composition, comprising an antibody, a nucleic acid molecule, a vector or an inhibitor of the present invention, and possibly a pharmaceutically acceptable carrier. These pharmaceutical compositions containing, for example, an inhibitor or pharmaceutically acceptable salts thereof, may conveniently be administered by any of the routes commonly used for drug administration, for example, orally, topically, parenterally, or by inhalation. Suitable salts include acetate, methyl ester, hydrochloride, sulfate, chloride, and the like. The compounds can be administered in conventional dosage forms made by combining drugs with standard pharmaceutical carriers according to conventional methods. These techniques may include mixing, granulating, and compressing or dissolving the ingredients, as appropriate, to form the desired formulation. It should be understood that the form and nature of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is combined, as well as with the introduction and other well-known variables. The carrier (s) must (s) be “acceptable” (and) in the sense of being compatible with the other ingredients of the drug and harmless to the recipient. The pharmaceutical carrier employed may be, for example, solid or liquid. Examples of solid carriers are lactose, magnesia, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and the like. Examples of liquid carriers are phosphate buffered saline, syrup, oil, such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, etc. Similarly, the carrier or diluent may include a material that slows the release well known in the art, for example glyceryl monostearate or glyceryl distearate, alone or in combination with wax.

The dosage regimen of the drug will be determined by the attending physician and other clinical factors, preferably in accordance with any of the methods described above. As is well known in medicine, dosages for any patient depend on many factors, including the size of the patient, the surface area of the body, the age, the specific compound to be administered, the gender, the time and route of administration, general health, and other medicines that are administered simultaneously. Monitoring progress is carried out by periodic surveys.

In addition, the use of pharmaceutical compositions containing antisense oligonucleotides specifically hybridizing with RNA, which are encoded by mutated variants of the ΝΒ81 gene according to the invention, contain antibodies that specifically recognize the mutated ΝΒ81 protein, but do not recognize or essentially do not recognize the wild-type functional form, possibly in cases in which the concentration of the mutated form in the cells must be reduced.

In addition, the present invention relates to a diagnostic composition or to a kit comprising any of the above polynucleotides, vectors, host cells, variants of proteins ΝΒ81, antibodies, inhibitors, nucleic acid molecules or corresponding vectors of the invention, and possibly suitable means for detection.

The kit of the invention may contain additional ingredients, such as selective markers and components for selective media, suitable for the production of transgenic cells and animals. The kit according to the invention can preferably be used to carry out the method according to the invention, and it can also, among other things, be used for various applications, for example in the field of diagnostics or as a research tool. Parts of the kit of the invention may be packaged individually in vials or in combination in containers or devices from a plurality of containers. The production of this kit is preferably carried out according to standard procedures known to those skilled in the art. A kit or diagnostic compositions can be used in methods for detecting expression of the mutant form of the gene ΝΒ81 and in any of the above methods of the invention, using, for example, immunoassay techniques such as radioimmunoassay or enzymatic immunoassay, or, preferably, nucleic acid hybridization and / or amplification techniques, such as those described above and in the examples, or microarray based techniques.

Some genetic changes lead to an altered conformational state of the protein. Restoring the normal or regulated conformation of mutated proteins is the most elegant and specific way to correct these molecular defects, although it is difficult. Pharmacological manipulations, therefore, may be aimed at restoring the conformation of the wild-type protein. Thus, polynucleotides and encoded proteins of the present invention can

- 10 011608 but also used for the design and / or identification of molecules that are capable of activating the function of the wild-type gene or protein ΝΒ81.

In another embodiment, the present invention relates to the use of a drug or prodrug for the manufacture of a pharmaceutical composition for treating or preventing a disorder diagnosed by the method described above.

In addition, the present invention relates to the use of an effective dose of a nucleic acid sequence encoding a functional and expressed wild-type protein # 81 for the preparation of a pharmaceutical composition for treating, preventing and / or slowing down a disorder diagnosed by the method of the invention. The gene encoding the functional and expressed protein ΝΒ81 can be introduced into cells, which, in turn, produce the protein of interest. Gene therapy, which is based on the introduction of therapeutic genes into cells using ex-νίνο or ίη-νίνο methods, is one of the most important applications of gene transfer. Vectors and methods suitable for gene therapy ίη-νίίτο or ίη-νίνο are described in the literature and are known to those skilled in the art; see, for example, Οίοτάαηο, No. 1, Meiuše 2 (1996), 534-539; 8yyaret, S1gs. Century. (1996) 911-919; Ληώ ^ Γκοη. 8s1epse 256 (1992), 808-813; 1kpeg Hans! 348 (1996), 370-374; MiYaikeg, ATP. Century. 77 (1995), 1077-1086; And apa, HIpe Meijushe 2 (1996), 714-716; AO 94/29469; AO 97/00957 or 8syaret, Siggey Θρίηίοη ίη ο ^ 1ιηο1οβ \ · 7 (1996) 3 635-640, as well as sources cited in this document. The gene can be designed for direct introduction or introduction through liposomes or viral vectors (for example, adenoviral, retroviral) into the cell. Preferably, said cell is a germ cell, an embryonic cell or an egg cell or a derivative thereof, most preferably said cell is a stem cell.

As is clear from the above, it is preferable that when used in accordance with the invention, the nucleic acid sequence is functionally linked to regulatory elements that allow expression and / or directing protein ΝΒ81 to specific cells. Suitable gene delivery systems that can be used in accordance with the invention may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors, such as, for example, herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses. For gene therapy, the delivery of nucleic acids to a specific site in the body can also be accomplished using a biolistic delivery system, such as described by Ayyashk (Proc. IAI. Ls. 8c1. ΫδΆ 88 (1991), 2726-2729). Standard methods for transfecting cells with recombinant DNA are well known to specialists in the field of molecular biology, for example, they are described in AO 94/29469, see also above. Gene therapy can be accomplished by directly introducing the recombinant DNA molecule or vector of the invention to the patient or by transfecting the cells with a polynucleotide or vector of the invention ex ν νο and infusing these transfected cells into the patient.

In a preferred embodiment of the use and methods of the invention, the disorder is cancer, as mentioned above.

Other ways of using the polymorphisms described in connection with the present invention, as well as means and methods that can be used in accordance with the above described embodiments, can be found in the prior art, for example, they are disclosed in ϋδ-Ά-5856104, where the means and methods used in forensic medicine, for paternity testing, correlation of polymorphisms with phenotypic traits, genetic mapping of phenotypic traits, etc., can be equally implemented in accordance with the present invention.

In addition, the present invention proposes a method for detecting susceptibility to prostate cancer in a subject, including detecting a change in the sequence of the г81 gene in a biological sample from a subject, where this change is an indicator of susceptibility to prostate cancer. The subject can be a person, for example, of Slavic origin.

In addition, the present invention provides a method for detecting a susceptibility to breast cancer in a subject, including detecting a change in the sequence of the г81 gene in a biological sample from a subject, where this change is an indicator of susceptibility to breast cancer. In some embodiments of the invention, breast cancer is an invasive breast cancer of a lobular subtype. The subject can be a person, for example, of Slavic origin.

In some embodiments of the invention, the change is an inherited change, for example 657e15. This change may be present in the sequence of one allele of the gene ΝΒ81, or this change may be present in the sequence of two alleles of the gene ΝΒ81. The change may be a mutation in the ΝΒ81 gene, for example, a mutation caused by an insertion into the gene, a deletion of the gene, or a replacement of the nucleotide (s) in the gene. In some embodiments, a change in the gene affects the production of the protein encoded by the gene 81, for example, inhibits it. The result of this change may be the production of a different, for example truncated, protein as compared with the protein that must be produced by the ΝΒ81 gene. Such a protein may not have the functional abilities that the protein encoded by the ΝΒ81 gene possesses.

In some embodiments, the change can be detected by PCR with allele-specific

- 11 011608 ny oligonucleotides (A80. A11e1e-zresb1s1scheyoperoRbe). assays for the detection of polymorphism of single-stranded conformation (88СР. 51пд1е-51гпбеб соГогтабопро1утотрЗзт). direct sequencing. microchips. direct sequencing. allele-specific amplification (А8А. a11е1е-зресб1с атр1Шсайоп) or КБЬР-PCR. Predisposition can be an inherited predisposition. In some embodiments, the biological sample may be a tissue sample. such as blood. and the biological sample may include leukocytes. In some embodiments of the invention, breast cancer is an invasive breast cancer of a lobular subtype.

Further. The present invention provides a diagnostic kit for identifying susceptibility to breast cancer or prostate cancer in a subject. comprising a packaging material and at least two different polynucleotides. able to amplify. at least. region of the gene ΝΒ81. In some embodiments of the invention, the amplified region includes a mutation 657b15. In some embodiments of the invention, the kit may contain polynucleotides No. zh6G. No. zh6t and zzbe15. The kit may also contain instructions. for example, instructions on how to use the kit to identify a predisposition to breast cancer or prostate cancer in a subject.

Ways and sets. suggested here. useful for detecting predisposition to cancer. such as prostate cancer and lobular invasive breast cancer. and they may also be useful for diagnosing cancer. such as prostate and breast cancer in the earliest clinical stages.

Change in gene ΝΒ81. for example, change 657be15. can be detected using any analysis. available to a person skilled in the art. which is capable of detecting change. eg. using nucleotide elongation analysis. sequence analyzes. hybridization assays and / or amplification assays. Change can be detected by testing for any form of DNA or RNA. received from the subject. For example. A person skilled in the art can identify the change. using PCR with allele-specific oligonucleotides. assays for the detection of polymorphism of single-stranded conformation (88SR. sshd1e-z1gapbeb sopottayopop1potrIzt). direct sequencing. allele-specific amplification. allele-specific hybridization and / or polymorphism of the lengths of restriction fragments after PCR amplification (CRPPCR). Hybridization can be carried out in different conditions. selected by the person skilled in the art. Some examples are described here below.

“Severe hybridization conditions” and “stringent hybridization washing conditions” in the context of polynucleotide hybridization experiments. such as Southern and Northern blot hybridization. depend on the sequence and differ in different environmental parameters. Longer sequences specifically hybridize at higher temperatures. "Tm" means temperature (at a certain ionic strength and pH). wherein 50% of the target sequence is hybridized with a highly appropriate probe. Specificity is typically a function of washing off after hybridization. and the critical factors are the ionic strength and temperature of the final wash solution. For DNA-DNA, Tt hybrids can be approximated on the basis of the equation MeshkoSh and No.aY. Apa1. Wusyet .. 138: 267 (1984); TT 81.5 C + 16.6 (1 M) +0.41 (% OS) 0.61 (% Gogt) 500 / b; where M is the molarity of monovalent cations. % OS - the percentage of nucleotides of guanosine and cytosine in DNA. % Gogt is the percentage of formamide in the hybridization solution and b is the length of the hybrid in base pairs. Tm is reduced by about 1 ° C for every 1% of non-pairing; in this way. Tm hybridization and / or washing conditions can be adjusted to hybridize the sequences of the desired identity. For example. if sequences with an identity of> 90% are assumed. TT can be reduced by 10 ° C. Usually. severe conditions are chosen as the temperature of about 5 ° C below the melting point (Tm) for a particular sequence and its complement at a certain ionic strength and pH. However, under very harsh conditions, it is possible to carry out hybridization and / or washing at a temperature of 1. 2. 3 or 4 ° C below the melting point (Tm); under moderately severe conditions, it is possible to carry out hybridization and / or washing at a temperature of 6. 7. 8. 9 or 10 ° С below the melting point (Tm); under conditions of weak stringency, hybridization and / or washing can be carried out at a temperature of 12. 12. 13. 14. 15 or 20 ° С below the melting point (Tm). When using the above equation. formulations for hybridization and washing and the desired T, the ordinary person skilled in the art will understand. that variations in stiffness of hybridization and / or wash solutions. naturally. described in the literature. If the desired degree of mismatching results in T less than 45 ° C (aqueous solution) or 32 ° C (formamide solution). it is preferable to increase the concentration of 88C so. to use a higher temperature. An extensive guide to the hybridization of polynucleotides can be found in Trzzep. Lover! From TesNsshschez w VyusNe1shzingu ab Motsiigiag Vyukoda NORSPH / aRop \ νίΐ1ι №.1s111 As1b Rgoez. Paradise I syar! em 2 Oueg \ ze \\ og rpps1r1ez oG Nyg1n1haIop upb not z1g oto gor1upisteyoy pro rugo azzauz. E1ze \ zeg. No. \ ν Wogk (1993). Usually. strict conditions are chosen as follows. that the temperature was about 5 ° C below the melting point (Tm) for a given sequence at a given ionic strength and pH.

An example of high hardness washing conditions is 0.15M # 1C1 at 72 ° C for about 15 minutes. An example of harsh washing conditions is washing at 0.2X 88 ° C at 65 ° C for 15 minutes (see the description of buffer 88C in Motessiag S1Oshid: A bhaota! Ot mapi1; 8tggok e! A1. 3gb eb. . (2001)). Often, high hardness washing is preceded by low hardness washing.

- 12 011608 to remove the background signal of the probe. An example of washing the medium hardness for a duplex, for example, more than 100 nucleotides is IX 88C at 45 ° C for 15 minutes. An example of a low hardness washing for a duplex, for example, more than 100 nucleotides is 4-6X 88C at 40 ° C for 15 minutes. For short probes (eg, from about 10 to 50 nucleotides) severe conditions typically include salt concentrations of less than about 1.5 M, more preferably from about 0.01 to 1.0 M, the concentration of sodium ion (or other salts) at pH from 7 , 0 to 8.3, and the temperature typically is at least 30 ° C and at least about 60 ° C for long probes (eg,> 50 nucleotides). Tough conditions can also be achieved by adding destabilizing agents such as formamide. As a rule, the signal-to-noise-background ratio of a 2X (or higher) signal, which is observed for an inappropriate probe in a particular hybridization analysis, shows the detection of specific hybridization. Polynucleotides that do not hybridize to each other under harsh conditions are still essentially identical when the proteins they encode are essentially identical. This happens, for example, when a copy of a polynucleotide is created with the maximum codon degeneracy allowed by the genetic code.

Very stringent hybridization conditions are chosen as equal to Tt for a particular probe. An example of stringent hybridization conditions for complementary nucleic acids, which has more than 100 complementary residues, on a Southern or Northern blot filter is 50% formamide, for example, hybridization is carried out in 50% formamide, 1M LaC1, 1% SDS at 37 ° C and washing is carried out in 0,1X 88C at a temperature of from 60 to 65 ° C. Approximate conditions of low stringency include hybridization with a buffer solution of from 30 to 35% formamide, 1M №С1. 1% SDS (sodium dodecyl sulfate) at 37 ° C and washing in 1X-2X 88C (20X 88C = 3.0M NaCl / 0.3M trisodium citrate) at a temperature of 50 to 55 ° C. Exemplary conditions of moderate stringency include hybridization in 40-45% formamide, 1.0 M NaC1, 1% SDS at 37 ° C and washing in 0.5X-1X 88 ° C at a temperature of 55 to 60 ° C.

Some polynucleotides that are useful for detecting changes are described here, and one skilled in the art will be able to construct other polynucleotides that will be useful in detecting a change. Thus, in the present invention also proposed polynucleotides containing any of E 0 III N0: 6-12, consisting essentially of these sequences or consisting of these sequences.

Human tumors are often associated with genomic instability, and the biochemical pathway for signaling DNA damage plays a critical role in maintaining the integrity of the genome in response to factors that damage DNA. This biochemical pathway may play an important role in the pathogenesis of prostate cancer. Transfer of the DNA damage signal is interrupted causing mutation syndromes discontinuity human chromosome, such as Nijmegen syndrome (ΝΒ8), Bloom syndrome, Fanconi anemia and ataxia telangiectasia (AT), which have a "spontaneous" chromosomal instability, immunodeficiency, and a predisposition to cancer ( E1deteb, 1993; and ΕιιΙηΕί e (a1., 2001). Genniy Nijmegen syndrome ΝΒ81 is mapped on chromosome 8c. | 21 and cloned (see Wagop e (a1. 1998; US Pat. No. 6458534; and SepBack, registration number АВ013139) Product gene ΝΒ81, nibrin (also called p95) is an integral component of the ΗΜΚΕ11 / ΚΚΑΌ50 / ΝΒ81 nuclease complex, which is part of the HXA1-associated genome control complex (BA8C), which is responsible for the repair of DNA damage (Eu (ac1 (a1., 2001)). The shortening deletion of 5n ... o in exon 6 of the gene ΝΒ81 was found in the vast majority of patients with ΝΒ 8. Most of the patients with 8, which were reported, are of Slavic origin and carry the founder mutation 657b15. This mutation is present with an unexpectedly high frequency of occurrence in Poland, Ukraine, and the Czech Republic (Wahupée et al., 2000). The role of gene ΝΒ81 in the development of prostate cancer and lobular invasive breast cancer has not yet been investigated.

As described in this document, it turned out that ΝΒ81 acts as a classic tumor suppressor gene, since biallel inactivation ΝΒ81 is observed in most tumors. However, some degree of haploid deficiency and the possible dominant-negative effect of mutations of ΝΒ81 are not excluded, since it has not been established that cells heterozygous for ΝΒ81 have an impaired ability to repair DNA. It is predicted that the founding allele ΝΒ81 results in a shortened protein of 219-754 amino acids (p26). The p26 lacks the critical domain required for interaction ΜΚΕ11. It is not known whether this mutant protein has any residual activity or exhibits a dominant negative effect. However, the 657be15 allele also creates an aberrant translation initiation site, which creates a partially functional variant of protein ΝΒ81 (p70). The p70 protein contains a ΜΚΕ11 binding domain, but does not provide a complete function within the ΜΚΕ11 complex. In light of this, it is possible that p70 can produce a dominant negative effect.

In addition, the present invention relates to the use of an inherited change in the sequence of the gene ΝΒ81 to identify a hereditary susceptibility to prostate cancer or invasive breast cancer of the lobular subtype in humans. Preferably, the specified inherited change is a mutation 657be15, and the person being studied is a person of Slavic origin. The presence of this inherited change can be detected by at least one method selected from Α8Θ PCR, 88СР, microchips, direct sequencing, Α8Α, КЕЬР PCR.

In addition, the present invention relates to a diagnostic kit for identifying a hereditary susceptibility to prostate cancer or invasive breast carcinoma of the lobular subtype by a PCR-based method that includes at least two different oligonucleotides that allow amplification of the region including the inherited change in the ΝΒ81 gene sequence where this amplified region is possible includes a mutation 657e15. Preferably, this kit contains primers No. of £ 6 £, No. of 166 and No. 15. The present invention also relates to a protocol for early detection of breast cancer, different from regular standards based on the incidence of the inherited mutation ΝΒ81.

These and other embodiments are described or apparent from the description and examples of the invention and are encompassed by them. Additional literature on any of the methods, applications, and compounds for use in accordance with the present invention can be found in public libraries using, for example, electronic devices and databases available on the Internet.

Pharmaceutical and diagnostic compositions, methods of use, methods according to the invention can be used to diagnose and treat all types of diseases, still unknown, related to variants of genes ΝΒ81 or dependent on them. The compositions, methods and uses of the present invention can and should be applied to humans, although the treatment of animals is also encompassed by the methods and uses described herein.

Brief description of the figures

FIG. 1 shows the LH analysis of microscopic MS tissue sections obtained from a proband of family 8 (lanes 1-5) and a proband of family 9 (lanes 1b-5b) using markers flanking the gene ΝΒ81 (lanes 1-3 and 1b-3b), and using exon-specific PCR (lanes 4-5, 4b-5b). The loss of the wild-type allele in cancer tissue is indicated by arrows. Dots indicate an allele with a founding mutation ΝΒ81.

FIG. 2 depicts the offspring of families with the mutation ΝΒ81. Segregation of the ΝΒ81 mutation with prostate cancer is shown in families 8, 9, 11, 12. The age of the individual is shown on the right side of each cancer case. A dot (·) in the lower right corner indicates that a blood sample was being analyzed. A plus (+) in the upper right corner of the symbol indicates the presence of a founding mutation ΝΒ81, and a minus (-) indicates relatives negative for this mutation. Arrows indicate probands. Completely black symbols mean cancer patients whose pathological results are available; 3/4 black characters indicate cancer patients for whom histopathology was not available. The type of cancer is indicated under each symbol. Additional family A, diagnosed at the Szczecin Cancer Heritage Center, does not belong to the groups of inherited MS.

FIG. 3 shows a fragment of the genomic sequence of the gene ΝΒ81, including exon 6. The sequence of exon 6 is shown in bold, and the mutation 657e15 is shown in italics (see also Sebyak, registration number АВ013139; Makita e (a1., 1998).

Hereinafter the invention will be described by biological examples, which are illustrative only and are not intended to limit the scope of the present invention.

Example 1. The establishment of a correlation between an inherited change in the sequence of the gene ΝΒ81 and a hereditary predisposition to prostate cancer or breast cancer by the example of analysis of the founding mutation 65715 in the gene ΝΒ81.

Relationship between 657e15 and prostate cancer

Patients.

Patients with suspected prostate cancer are hospitalized at the Urology Clinic in Szczecin. Criteria for suspected prostate cancer are elevated levels of Ρ8 4 - above 4.0 ng / ml, or detection of abnormalities in the study in the supine position. Prostate cancer is diagnosed in the clinic on the basis of a biopsy of 1 ° EC1. 'T with ultrasound monitoring performed in suspicious cases. Biopsy tissues stained using standard techniques were evaluated by pathologists at the Department of Genetics and Pathology in Szczecin. The final diagnosis was confirmed by one pathologist, Professor Jan Lubinski (ZapiMikI).

All 359 men diagnosed with prostate cancer at the University Hospital in Szczecin, Poland, between 1999 and 2002, were invited to participate in this study. Of these, 340 (95%) agreed to participate. All patients were recruited to participate in the study within 6 months from the date of diagnosis. Family histories of cancer were obtained from each subject. 35 patients (10.3%) had one or more first or second degree relatives with prostate cancer (family cases). The inventors also included a second group of 21 familial cases of prostate cancer from men who were sent to the Center for Cancer Heredity by family doctors or urologists for a study on combining prostate cancer family cases. There were a total of 56 family cases and 305 non-family cases. Randomly selected familial cases contained an average of 2.1 cases of prostate cancer (mean age of occurrence 67.3 years), and familial cases selected by the Inheritance Cancer Center contained 2.6 cases of prostate cancer (mean age of occurrence 63.3 years) . The number of unaffected controls was 1500. 1 thousand controls were selected.

- 14,011,608 randomly from a computerized list of patients of three family practices in Szczecin (508 women and 492 men; age range from 26 to 89 years). Additionally, a second control group of 500 non-selected newborns from Szczecin was included, for which umbilical cord blood samples were available. FIG. 2 depicts the offspring of families with the mutation ΝΒ81.

Relationship between 657bs15 and lobular cancer of lobular subtype

Patients.

The study group included 2012 women with breast cancer who were selected without considering the nature of the cancer. Breast cancer cases were recruited from 8 hospitals from all over Poland (Table 1). The patients were confirmed several times by the pathological departments of medical institutions. Only primary invasive breast cancer was included (cases of ES18 and HC18 were excluded). Patients were collected from 2002 to 2003. Two thousand controls from the general population were used to study the connection between the founding allele ΝΒ81 (1000 unselected adults from Szczecin and 1000 newborns in 2003 from six hospitals from all over Poland (Szczecin, Bialystok, Horzov, Katowice and Wroclaw). The group was rated for frequency ΝΒ81 657bs15 in the general population.

Table 1

The frequency of the mutant founder allele ΝΒ81 among patients with breast cancer in eight regions of Poland

City The number of subjects ΝΒΜ 657 <1e15 Szczecin 511 five Bielsko-Biala 172 2 Opole 480 five Poznan 258 one Krakow 251 2 Koszalin 110 one Lublin 179 one Bydgoszcz 51 0 Total 2012 17

Example 2. Detection of a mutation 657bs15 in the gene ΝΒ81.

The inventors used X8O-PCR and sequencing to detect the founder mutation ΝΒ81 in DNA isolated from peripheral blood leukocytes.

Received 5 ml of peripheral blood and mixed with 100 μl of 1M EDTA, then centrifuged in 50 ml of polypropylene tubes for 10 min at 3000 d at 4 ° C. The serum in the upper phase was removed and the precipitate containing the cells was mixed with 45 ml of 2X buffer (0.1 M ΝΗ ₄ Ο 1. 0.25 M KHCO ₃ , 1 mM EDTA) and left for 15 min at 4 ° C. Then the mixture was centrifuged at 3,000 d for 10 min at 4 ° C. The supernatant was removed after centrifugation. The remaining precipitate with leukocytes was suspended in 2X buffer and centrifuged for 10 minutes at 3000 ° C at 4 ° C. This cleaning of leukocytes in 2X buffer and centrifugation was repeated 3 times to obtain a pure leukocyte sediment. These leukocytes were mixed with 3 ml of Pivar buffer (50 mM IaC1, 25 mM MdC1 ₂ , 1 mM EDTA; pH 8.0) with 200 µl of 10% SDS and 500 µg of proteinase K. Perevar was performed for 24 hours at 37 ° С .

DNA was purified using phenol / chloroform. Briefly, the products were mixed with 3 ml of phenol, buffered with 0.5 M Tris HC1 (pH 8.4), and then with 3 ml of a mixture of chloroform and isoamyl alcohol (1:25 v / v). The mixture was shaken for about 1 min and centrifuged for 10 min at 8000 d at 20 ° C. After centrifugation, the upper phase was transferred to a new tube and mixed with an equal volume of chloroform, and then centrifuged for 10 minutes at 8000 d. The above cleaning with chloroform was repeated 3 times until the protein ring disappeared in the interphase.

The purified aqueous phase containing DNA was mixed with 5M # 101 in a ratio of 10: 1 (v / v) and 96% ethanol with respect to the aqueous phase from # 1C'1 to ethanol 1:10 (v / v). The mixture was left overnight at 20 ° C. The resulting precipitate DNA was placed in a new tube and purified with 70% ethanol, centrifuged at 3000 d for 5 min and the ethanol was poured. Then the purified DNA precipitate was dried in an open tube for 30 min at 37 ° C. DNA resuspended in 400 μl of TE buffer (25 mM Tris HC1, 1 mM EDTA; pH 8.4) was stored at 4 ° C until use.

Allele-specific PCR (L8O-PCR)

L8O-PCR reaction was carried out in the apparatus ΌΝΆ T11sgta1S'ue1sg 9600 (Retksh E1teg) in a volume of 25 l comprising 1 ul (50 ng) genomic DNA, 4 pmol primer №zeh6G, 6 pmol of primer №zeh6t, 10 pmol primer №zbe15 2 , 5 μl of PCR buffer (100 mM Tris-HC1, 500 mM KC1, 15 mM MdCl ₁ , 1 mg / ml gelatin; pH 8.6), 200 μM each 6LPE, 6STR, 6STR and 6TPR, and 1 unit. Tad DNA polymerase. In each reaction, 2 positive controls were used (controls with DNA from heterozygote ΝΒ81 and homozygotes ΝΒ81) and 2 negative controls (control DNA from the patient, negative for mutation ΝΒ81, and control without DNA).

Conditions L8O-PCR:

a) initial denaturation - 95 ° C, 5 min;

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b) 11 cycles, each of denaturation - 94 ° C, 30 s, annealing of primers - from 62 to 56 ° C, 30 s *, elongation of primers - 72 ° C, 30 s;

c) 30 cycles, each of denaturation - 94 ° C, 30 s, annealing of primers - 56 ° C, 30 s, elongation of primers - 72 ° C, 30 s.

* During the first 11 cycles, the annealing temperature of the primers was reduced by 0.6 ° C in each cycle, starting from 62 ° C in the first cycle and ending at 56 ° C in the eleventh cycle (detail: 1st cycle - 62 ° C, 2 the first cycle is 61.4 ° C, the 3rd cycle is 60.8 ° C, the 4th cycle is 60.2 ° C, the 5th cycle is 59.6 ° C, the 6th cycle is 59 ° C , The 7th cycle is 58.4 ° C, the 8th cycle is 57.8 ° C, the 9th cycle is 57.2 ° C, the 10th cycle is 56.6 ° C, the 11th cycle is 56 ° C).

The sequence of primers used in A8O-PCR:

Νόδθχ6Γ. 5 'SASSTTO ATOAASSATST DES) GO ΝΟ: 6) No. G6, 5' SSTTAASAASTASTATAATASASAS DES) GO ΝΟ: 7) No. Sbe15, 5 'SSACSSASSAAASAAATSTT (specific primer for 657be15) DEO GO load buffer and subjected to electrophoresis on agarose gel (1.5% agarose gel DeKesh RMS), 1X TBE buffer, 25 pg / ml ethidium bromide) at 6 V / cm for 30 minutes. Separated products are visualized in UV light. All cases in which an additional, shorter PCR product was observed was sequenced to confirm the presence of the founder 81 mutation.

Sequencing

Matrix PCR.

Exon 6 of gene ΝΒ81 was amplified with primers Hb5e6G and Hb5e6g under such conditions as described for A8O-RBC, with the only difference that primer No.§b15 was not used in the template PCR.

Purification of PCR products.

The amplification products of exon 6 were transferred with a pipette into the sample reservoir M1sgosoi-100 (Atsoi) placed in a vial, and 400 μl bN ₂ O was added to the tank and centrifuged at 1,850 d for 15 min. After centrifugation, the sample tank was placed in a new vial filled with 400 μl bN ₂ O and centrifuged at 1,850 d for 15 minutes. The last stage was repeated 3 times. The sample tank was placed upside down in a new vial, and then centrifuged for 3 minutes at 9000 d. All centrifugations were carried out at 25 ° C. Approximately 5 μl of the purified PCR product was visible in the vial, and it was diluted in 20 μl of bN ₂ O.

Sequencing PCR.

Asymmetric sequencing PCR was performed in an Oeye Atr RSC 8000 9600 (Regkt E1teg) thermocycler in a volume of 20 μl containing 1 pmol of primer No. 16G, 4 μl of purified PCR product, 8 μl of V | dGPTGTT1G Keabu Keasboi IGa Ia Ia Ia Ia Ia Ia Ie Ie, I & I, I & I, I & I Control System, and 17 μl B | O5U51et5). In addition, a sequencing reaction with primer No. Gullar was carried out to confirm the results with the direct primer.

Sequencing conditions:

initial denaturation - 96 ° C, 30 s;

cycles, each of denaturation - 94 ° C, 30 s, annealing of primers -56 ° C, 30 s, elongation of primers - 72 ° C, 30 s.

μl of the sequencing product was placed in a 0.5 ml Eppendorf tube, 60 μl of 96% ethanol and 2 μl of 3M sodium citrate (pH 4.6) were added. Samples were centrifuged for 20 minutes at 3,000 d at 25 ° C. Then the supernatant was removed and 200 μl of 70% ethanol was added to purify the precipitate. After 5 min of centrifugation at 3000 d at 25 ° C, the supernatant was removed. The precipitate was dried in YerreibogG SoiseiGalog 5301 for 20–30 min, and then resuspended in 4 μl of the deposition buffer (150 μl of deionized formamide, 50 μl of 50 mM EDTA, 0.05% Eeh1gap B1ie). Samples were denatured for 4 min at 94 ° C, placed on ice, and applied onto a denaturing polyacrylamide gel (4% 19: 1 polyacrylamide gel, 1X TBE, 6M urea). Electrophoresis was carried out in AB1 PK18M 377 ΩΝΛ 8thcieyseg (Arriebe B | o5-515). The data were collected and analyzed using the AB1 PK18M 377 SoPesbop 8op software and 8ecs / 8Appsd Aia1188 8th 8th Auger 3.0 (Arriebe Vy551515) software.

Example 3. Analysis of the loss of heterozygosity (LON) in prostate and breast cancer. Micro-dissection and DNA Extraction

To analyze whether the wild-type allele ΝΒ81 was lost in prostate cancer and breast cancer, the inventors conducted an LHON analysis in tumors after micro-dissecting carriers of mutation ΝΒ81 - in 9 cases of prostate cancer and 5 cases of breast cancer. LH was performed using markers flanking the gene ΝΒ81 - Ό8888, Ό881811 and primers labeled with a fluorescent label specific to exon 6 ΝΒ81 (9).

Slices were prepared in 5 μm of formalin-fixed and paraffin-embedded tissues and size

- 16 011608 spat them on the glasses. For each patient, tissue was cut for 6 glasses. One glass was stained with hematoxylin / eosin. The remaining glass used for microdissection. Sections were paraffin free in two xylene changes for 5 minutes. The sections were hydrated using a series of graded alcohols (in 96% ethanol (2 times), 70% ethanol and N ₂ O, each for 5 minutes). Glasses were stained with hematoxylin. Using a light microscope, homogeneous fields of cancer cells were selected in GE-stained sections. These fields were carefully micro-dissected using a needle from glasses stained with hematoxylin under a light microscope, avoiding contamination with benign cells. Sections obtained by microdissection were placed in 1.5 ml Eppendorf tubes. In parallel, normal tissues were cut from the same glasses and placed in other separate tubes.

Then the tissues subjected to microsection were placed in 1 ml of buffer for enzymatic hydrolysis (50 mM Tris-HC1, 1 mM CaC ₂ , pH 8.0) with 20 μl of 10% SDS and 500 μg of proteinase K. Negative controls were used in each series tissue. Enzymatic hydrolysis was carried out at 55 ° C for 2 weeks. On days 3 and 6 of the hydrolysis, an additional 100 μg of proteinase K was added. After hydrolysis, the proteinase was inactivated by heating at 96 ° C for 10 minutes. 500 μl of the product of enzymatic hydrolysis was purified in tubes M1sgosop-100 (Ash1sop) according to the above method. After purification, approximately 5 μl of the solution containing DNA was diluted in 50 μl of JN ₂ O.

BON analysis

LHON analysis was performed in 3 PCR reactions with fluorescent primers:

1) PCR1 with primers 1) 88881 '- 5' TCCA6CA6A6AA6666TTAT (8 EC) ΝΟ: 9); Ό8888γ - 5 ⁷ 66CAAA6A6AASSTSATSA6A (8EC) ΙΌ ΝΟ: 10);

2) PCR2 with primers Ό881811Γ - 5 'СССАСССССААААТС (8 ЕС) ΝΟ: 11); Ό881811Γ - 5 '666ТТТА666АА6Т6СА6АА (8ЕС) ΙΌ ΝΟ: 12);

3) PCR3 with primers No.k6G and Ixx6g flanking exon 6 ΝΒ8 1. containing 657e15. The PCR reaction was carried out in an ECA TilengaSluster 9600 (Regksh E1teg) in a volume of 25 μl, which included 4 μl of DNA isolated from tissues, 2.5 μl of PCR buffer (100 mM Tris-HCl, 500 mM KC1, 15 mM MdCl ₂ , 1 mg / ml of gelatin; pH 8.6), 200 pM of each YATR, YSTR, YSTR and YTTP, 1 unit. Tad DNA polymerase and 10 μg bovine serum albumin (BSA - EgsheShak). A mixture of PCR1 included an additional 5 pmol of the Ό8888Γ primers and Ό8888γ, PCR2 - 5 pmol of the Ό881811Γ and Ό881811Γ primers, and PCR3 - 5 pmol of the No.Kh6G and HKh6H primers. Positive and negative controls were used in each PCR reaction.

PCR conditions:

c) initial denaturation - 95 ° C, 5 min;

d) 42 cycles, each of denaturation - 94 ° C, 30 s; annealing of primers -56 ° C, 30 s; primer elongation - 72 ° C, 30 s.

µl of the PCR product was diluted in 10 µl of the deposition buffer (150 µl of formamide, 50 µl of 50 mM EDTA, 0.05% EEC1). After denaturation for 4 min at 94 ° C, the samples were placed on ice and applied to a denaturing polyacrylamide gel (4% 19: 1 polyacrylamide gel, 1X TBE, 6M urea). Electrophoresis was carried out in ΆΒΙ RSH8M 377 ΌΝΛ 8es. | Yepseg (Arrieu Byukuyetk). The data were collected and analyzed using the software ΆБI РК18М 377 Со1ес1юп 8оП \ гаге and Sep8sap Ap1uk1к 8оП \ гаге Уегаоп 3.0 (ArrNey E | Okuk1tk). A decrease in the signal in one allele by at least 70% was taken as the recognition threshold for ΕΟ4.

Statistical analysis was performed using Chi-square test.

Mutation ΝΒ81 was present in 9 out of 340 randomly selected cases of prostate cancer (2.6%) compared with only 9 out of 1500 (0.6%) control individuals from the general population (ratio oyk = 4.5; 95% 0 = 1, 7 to 11.5; p = 0.002). The inherited mutation 657e15 was present in 5 of 56 (9%) family cases (ratio oyk = 16; 95% 0 = 5.2 to 50; p <0.0001). The inventors investigated the segregation of the ал81 mutant allele with prostate cancer in four families. The inventors were able to establish the state of mutation in two affected men from each family. In each family, a mutation ΝΒ81 was present in both affected members.

Allele 657e15 was found in 17 (0.8%) of 2012 tracked breast cancers compared with 8 in 2000 controls (ratio oyk = 1.9; 95% 0 = 0.8 to 4.2, p = 0.17 ). Paraffin-embedded tissues received from 12 cases of breast cancer from carriers mutations ΝΒ81. The type of these tumors was determined by two pathologists after staining with hematoxylin / eosin (H / O) and immunohistochemical staining. 9 out of 12 cases of breast cancer were large cell infiltrating lobular carcinomas (see Table 2). D / E slices were available from 491 of 2012 cases investigated. All these cases were from the same center: the district cancer hospital in Szczecin. Two pathologists established a type of breast cancer in all 491 cases. Lobular carcinomas were diagnosed in 53 of the 491 breast cancers observed. Of these 53 patients with breast cancer, mutations ΝΒ81 were detected in 4 (7.5%) cases (ΟΚ 18.4, 95% 0 = 5.5-62, p <0.0001). Thus, the ΝΒ81 mutation is associated with large cell infiltrating lobular carcinoma of the mammary gland.

- 17 011608

table 2

Histopathological data of carriers of change 657b15 among women with breast cancer

Pyacien t Age (years) The first pathological diagnosis Pathological diagnosis after re-evaluation of glasses one 44 Infiltrative ductal carcinoma Infiltrative ductal carcinoma 2 56 Infiltrating lobular carcinoma Infiltrating lobular carcinoma 3 38 Infiltrative ductal carcinoma Infiltrative ductal carcinoma four 42 Infiltrative ductal carcinoma Infiltrating lobular carcinoma five 47 Infiltrating lobular carcinoma Infiltrating lobular carcinoma 6 47 Infiltrating tubular carcinoma Infiltrating tubular carcinoma 7 68 Infiltrating lobular carcinoma Infiltrating lobular carcinoma eight 46 Infiltrating lobular carcinoma Infiltrating lobular carcinoma 9 57 Infiltrating lobular carcinoma Infiltrating lobular carcinoma ten 49 Infiltrating ductal Infiltrating lobular carcinoma carcinoma eleven 46 Infiltrating lobular carcinoma Infiltrating lobular carcinoma 12 52 Infiltrating lobular carcinoma Infiltrating lobular carcinoma

Loss of the ΝΒ81 allele was observed in 7 of 8 cases of prostate cancer (Fig. 1) and 5 of 5 cases of breast cancer. Data on the loss of heterozygosity suggest that the functions of ΝΒ81 are the classic functions of the tumor suppressor gene. Loss of heterozygosity in the ΝΒ81 locus is also indicated for ovarian cancer and malignant melanoma (8, 10). Clinically, Nijmegen syndrome is a recessive genetic condition. A heterozygous state can be harmless at the cellular level, but the loss of heterozygosity makes cells hemizygous for the mutant allele. Cultured cells that are homozygous for the ΝΒ81 mutation are prone to chromosomal aberrations (10).

The founding allele ΝΒ81 is found to be responsible for about 1 out of 11 families with two or more than two cases of prostate cancer in Poland. Based on the relative risk of 4.5 and the mutation frequency of 1 per 167, the inventors estimate that this gene is responsible for approximately 2% of prostate cancers in this country. The inventors did not observe a statistically significant excess of the allele of the founder ΝΒ81 in women with breast cancer who were selected without taking into account the nature of the cancer. However, 657be15 was associated with an increased risk of lobular carcinoma of the mammary gland (0B 18.4, p <0.0001). Considering the geographical distribution of the described clinical cases of Nijmegen syndrome, the 657be15 mutation can also make an important contribution to prostate cancer and lobular breast cancer in patients of Slavic origin from other countries (allele 657be15 is responsible for all cases of the Nijmegen syndrome in all Slavic populations described to date) .

Thus, the diagnosis of these types of cancer, especially in this ethnic group, can be facilitated by using simple A80-PCR analysis for the main founding mutation ΝΒ81. Diagnosis of prostate and breast cancer in other non-Slavic populations can be improved in a similar way.

The risk of prostate cancer is approximately 3% of the population of Poland with 40 million people. Considering the frequency of 2.6% of the founding mutation ΝΒ81 in subjects with prostate cancer, approximately 15,000 carriers of ΝΒ81 will develop prostate cancer, which is approximately 14% of all men affected by the founding mutation ΝΒ81. Since there was a significant difference in the frequency of mutation 657b15 in familial cases versus tracked cases (ratio of cbm = 16; 95% C1 = 5.2 to 50; p <0.0001), the risk of prostate cancer is higher if a relative of the carrier 81 is afflicted with cancer prostate

This is also the first report, which shows that the ΝΒ81 mutation most likely predisposes to the lobular subtype of invasive breast cancer. The risk of invasive lobular breast cancer is approximately 0% among the 20 million female population of Poland. Considering the incidence of 8% of the founding allele у81 in subjects with lobular invasive breast cancer, approximately 8,000 female carriers of ΝΒ81 will develop breast cancer, which is approximately 8% of women with a founding mutation ΝΒ81.

The average age of prostate cancer and breast cancer diagnosis is 68 and 58 years, respectively.

- 18 011608

The latter is significantly lower (50 years) for breast cancer diagnosed among carriers of mutation ΝΒ81. Consequently, mammography for women who are positive for mutation начин81 should be started earlier, that is, probably not later than at the age of 40.

Links

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2. Ri1ak1, Μ., Bsh, ΕΜ. Sygothokots lgkadads kupbgotsk apb 111s BKSA1 dspots kshussShaps comotr. Tsspbk ΜΘ1. Μ ^., 7; 560-565, 2001.

3. Uagop, K., with! a1., Nypp, and Potts1 ΟΝΛ battles1c-kyapb £ ssak gsrai rgo! bc, 18 ti1a1sb ws outtstspc. Cc11, 93: 467-76, 1998.

4. Satsu, BR., With! a1., Tys ys ^ s11 / y Kab50 rgo! ssot sotr1skh apb Yutsdsp Ushkads kupbgotts: ypkads oG baty1k-ky! gapb legsak gsrai! s! ss11i1ag BIA batads gsropks. Cc11, 93: 477-86, 1998.

5. Uagop, K., with! A1., Syshs1 Aksspa1pstsp! og Üutsdsp bargains kupbgotts (ΝΒ8) appbstg1sprsso og! ys ta) ogi! iop, 657bs15, w 11zgsss 81at ar1a! yupk. Eig I. Neath. Ssss !., 8: 900-902, 2000.

6. §SStapot, E. An tsgsaksb pkk Gog Tidepap! psor1akt sh ys! sgo7udo ck Gog and kupbgotts og t1sgossryyau, pohta1 t! sShssps, dgoMy gs1agba11op, gstagkaHs Haask, ttspoppspsypsu appbigototokot1 tk1ay11yyu. ΜιιΐΐΙ. Ksk., 238: 321-4, 1990.

7. Sogkk1, V., with! a1., Ссгтыпс 657бс15 т! айоп ш! сс ΝΒ81 дссс т Ьгсак! Sapssg Risp! 1p !. I. Sapsssg, 106: 379-81, 2003.

8. Bsshak, T., c! a1., I. Ssgtkps 657bs15 tSharop t! ys ΝΒ81 dsps t raysp! to \\ Y11 taydpap! ts1apota og! yc kkt. Μс1апота Кск., 13: 365-370, 2003.

9. Sui1kkz, S., with! a1., ΝΒ81 1k and rgok! and! with sapssg kikssryyyu dsps. Sapsss Ksk. 2004 Pcb. 15; 64 (4): 1215-9.

10. νап бсг Бигд! with! a1., Highlights bccads kupbgotts. I. Μsb. Ssps!, 33: 153-6, 1996.

11. Rykyuska-NaBaka, I. s! a1., Utsdsps bgsakads kupbgotts dpsp (ΝΒ81) a1 (sga (first apk ik rgo! ssh (tbpp) skrgskkyup 1p yitap otapap! itoigk. App. Neath. Spsi!., 66: 353-9, 2002.

Claims (53)

CLAIM 1. A polynucleotide associated with an increased hereditary predisposition to prostate cancer or invasive lobar cancer of a lobular subtype, selected from the group consisting of: (a) a polynucleotide encoding the molecular variant of the ΝΒ81 polypeptide, wherein said polynucleotide contains a nucleotide replacement, a nucleotide deletion, an additional a nucleotide or an additional nucleotide and substitution of a nucleotide at a position corresponding to the position of 18155, 18156, 18157, 18158 or 18159 of the ΝΒ81 gene (registration number in Cspbapk: AB013139); (b) a polynucleotide encoding the molecular variant of the ΝΒ81 polypeptide, wherein said polynucleotide contains a deletion of the nucleotide at the position corresponding to the positions 18155, 18156, 18157, 18158 or 18159 of the ΝΒ81 gene (registration number in Cbbapk: AB013139); (c) a polynucleotide encoding a molecular variant of the ΝΒ81 peptide, wherein said polypeptide has a deletion of an amino acid at the position from 234 to 754 of the ΝΒ81 polypeptide (8ЕО Ш ΝΟ: 2) and, possibly, at least one substitution selected from replacing Buk by Akp in positions 219, С1п on bci in position 220, 11с on С1п in position 221, Рис on Агд in position 222, Beech on С1и in position 223, С1у on Akp in position 224, Beech on 11с in position 225, Tyg on Tug in position 226, Rice for 11s in position 227, 11s for Rice in position 228, Rice for C1i in position 229, Bsi for Suk in position 230, ACP for C1p in position 231, A1a for Tyg in position 232, B to at A1a at position 233 of the polypeptide ΝΒ81 (8EO W ΝΟ: 2); (d) a polynucleotide encoding a molecular variant of polymeptide ΝΒ81 having the amino acid sequence 8EO W ΝΟ: 4. 2. The polynucleotide according to claim 1, where the result of a nucleotide deletion, insertion and / or substitution is an altered expression of a variant of the ΝΒ81 gene compared to the corresponding wild-type gene. 3. The vector containing the polynucleotide according to claim 1 or 2. 4. The vector according to claim 3, where the polynucleotide is functionally linked to regulatory expression sequences that allow expression in prokaryotic or eukaryotic cells. 5. A host cell obtained by genetic engineering, containing the polynucleotide according to claim 1 or 2 or the vector according to claim 3 or 4. 6. A method of producing a molecular variant of protein ΝΒ81 or its fragment associated with an increased hereditary predisposition to prostate cancer or invasive lobar cancer of the lobular subtype, wherein: (a) the host cell is cultured according to claim 5 and (b) the protein is isolated or fragment from the culture. 7. A method of producing cells capable of expressing a molecular variant of the ΝΒ81 gene associated with an increased hereditary predisposition to prostate cancer or invasive breast cancer of a lobular subtype, comprising genetically engineered cells containing the polynucleotide according to claim 1 or 2 or the vector according to claim .3 or 4. 8. Protein ΝΒ81 or its fragment associated with an increased hereditary predisposition - 19 011608 to prostate cancer or invasive breast cancer of a lobular subtype, which is encoded by the polynucleotide according to claim 1 or 2 or obtained by the method according to claim 6 or from cells obtained by the method according to claim 7. 9. An antibody that specifically binds to the protein of claim 8. 10. The antibody according to claim 9, which specifically recognizes an epitope containing one or more amino acid substitutions, as defined in claim 1 or 2. 11. The nucleic acid molecule complementary to the polynucleotide according to claim 1 or 2. 12. A nucleic acid molecule capable of specific recognition and cleavage of a polynucleotide according to claim 1 or 2. 13. A vector containing a nucleic acid molecule according to claim 11 or 12. 14. A transgenic animal other than human, containing at least one polynucleotide according to claim 1 or 2, or a vector according to claim 3 or 4. 15. A transgenic animal other than human, according to 14, additionally containing at least one inactivated wild-type allele of the gene ΝΒ81. 16. A transgenic animal other than human, according to 14 or 15, which is a mouse or rat. 17. A method for identifying and producing a ΝΒ81 modulator capable of modulating the activity of a molecular variant of the ΝΒ81 gene or its gene product, wherein: (a) the protein of claim 8 or a cell expressing the molecular variant of the ΝΒ81 gene containing the polynucleotide according to claim 1 are brought into contact or 2, in the presence of components capable of giving a detectable signal in response to the activity of protein ΝΒ81, with the compound to be screened, under conditions that allow the activity of protein ΝΒ81 to occur, and (b) establish the presence, absence of signal or increase Igna generated protein activity ΝΒ81, wherein the presence or increase of the signal is indicative of the presence of a putative modulator. 18. The method according to 17, where the specified cell is a cell according to claim 5, obtained by the method according to claim 7 or contained in a transgenic animal other than humans, according to any one of paragraphs.14-16. 19. A method for identifying and producing a ΝΒ81 modulator capable of modulating the activity of a molecular variant of the ΝΒ81 gene or its gene product, in which: (a) the protein of claim 8 is contacted with the first molecule, which is known to bind to the ΝΒ81 protein, to form a first complex comprising said protein and said first molecule, (b) said first complex is brought into contact with the compound to be screened, and (c) it is determined whether said compound is displacing said first molecule from said first complex. 20. The method of claim 19, wherein said step of determining comprises determining the formation of a second complex of said protein and said compound. 21. The method according to claim 19 or 20, wherein said step of determining comprises determining the amount of said first molecule unrelated to said protein. 22. The method according to any one of claims 19-21, wherein said first molecule is one of the parts of the nMKE11 / yKAP50 / HB81 nuclease complex. 23. The method according to any one of claims 19-22, wherein said first molecule is labeled. 24. A method for diagnosing an increased hereditary predisposition to prostate cancer or invasive breast cancer of a lobular subtype, in which: (a) determine the presence of a polynucleotide according to claim 1 or 2 in a sample taken from a subject, and / or (b) determine the presence of protein by item 8. 25. The method according to paragraph 24, comprising carrying out a polymerase chain reaction (PCR), ligase chain reaction, restriction enzymatic hydrolysis, direct sequencing, nucleic acid amplification procedures, microarrays, hybridization, or immunological analyzes. 26. The method according to any one of paragraphs.24-25, in which the subject is additionally administered a drug to destroy or weaken said cancer. 27. The method according to any one of paragraphs.24-26, further comprising introducing into the cells: (ί) a wild-type functional and expressed gene ΝΒ81 or (ίί) a nucleic acid molecule according to claim 11 or 12 or a vector according to claim 13. 28. A method of preparing a pharmaceutical composition comprising the steps of a method according to any one of claims 17-23 and (c) synthesizing a compound identified and obtained in step (b), or a derivative thereof and / or preparing a preparation therefrom in a pharmaceutically acceptable form. 29. A method of manufacturing a pharmaceutical composition in which a drug or prodrug is prepared in a form suitable for the therapeutic use and prevention or amelioration of cancer in a subject who is diagnosed with the method of claim 24. 30. The method according to p. 28 or 29, where the specified compound-drug or prodrug is a derivative of the drug specified in p. 31. An inhibitor identified or obtained by the method according to any one of paragraphs.17-23. 32. The inhibitor of claim 31, which specifically binds to the protein of claim 8. 33. An oligonucleotide for genotyping individual ΝΒ81 alleles, wherein said oligonucleotide is about 15 to 50 nucleotides in length and contains a nucleotide sequence included in one of 8EO W ΝΟ: 1, 8EO W ΝΟ: 3, 8EO W ΝΟ: 5-8EP W ΝΟ: 12, or after - 20 011608 completeness complementary to any of them. 34. A method for detecting a predisposition to prostate or breast cancer in a subject, the method comprising: (a) detecting in a biological sample taken from a subject, a change in the sequence of the ΝΒ81 gene, where said change causes a loss of the function of the ΝΒ81 gene; and (b) linking the change with a predisposition to prostate cancer or breast cancer in a subject, thereby detecting said predisposition. 35. The method according to clause 34, where the change is an inherited change. 36. The method according to clause 34, where the inherited change is a change of 657be15. 37. The method according to clause 34, where the subject is a human. 38. The method according to clause 34, where the person is of Slavic origin. 39. The method according to clause 34, where the change is detected by PCR with allele-specific oligonucleotides (L8O-PCR), tests for the detection of single-stranded conformation polymorphism (88СР), direct sequencing, allele-specific amplification (L8L), microarrays or PCR with using restriction fragment length polymorphism (KBR-PCR). 40. The method according to clause 34, where the specified predisposition is hereditary. 41. The method according to clause 34, where the biological sample is a tissue sample. 42. The method according to paragraph 41, where the tissue sample is a blood. 43. The method according to clause 34, where the biological sample contains white blood cells. 44. The method according to clause 34, where the breast cancer is an invasive breast cancer lobular subtype. 45. The use of an inherited change in the ΝΒ81 gene sequence to detect a hereditary predisposition to prostate cancer or breast cancer in a subject. 46. The application of claim 45, wherein said inherited change is a 657be15 mutation. 47. The use of claim 45, wherein the subject is a human. 48. The application of clause 47, where the person is of Slavic origin. 49. The use of claim 45, wherein the breast cancer is an invasive lobar cancer of the lobular subtype. 50. The use according to any one of claims 45-49, wherein the presence of an inherited change is detected using at least one of the methods selected from L8O-PCR, 88CP, microarrays, direct sequencing, L8L or KPP-PCR. 51. A diagnostic kit for identifying a predisposition to breast or prostate cancer in a subject, comprising packaging material and at least two different polynucleotides, allowing amplification of at least a portion of the ΝΒ81 gene. 52. The kit of claim 51, wherein the amplifiable portion contains a 657be15 mutation. 53. The kit according to claim 51, containing polynucleotides No.§ex6 £ (8EC ΙΌ ΝΟ: 6), No. 8ech6g (8EC ΙΌ ΝΟ: 7) and No.> 8be15 (8EC ΙΌ ΝΟ: 8).