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WO2008047128A2 - Matériaux et méthodes permettant de déterminer la susceptibilité au cancer - Google Patents

Matériaux et méthodes permettant de déterminer la susceptibilité au cancer Download PDF

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WO2008047128A2
WO2008047128A2 PCT/GB2007/003977 GB2007003977W WO2008047128A2 WO 2008047128 A2 WO2008047128 A2 WO 2008047128A2 GB 2007003977 W GB2007003977 W GB 2007003977W WO 2008047128 A2 WO2008047128 A2 WO 2008047128A2
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palb2
mutation
nucleic acid
mutations
cancer
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WO2008047128A3 (fr
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Nazneen Rahman
Michael Rudolf Stratton
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to materials and methods for determining a susceptibility to cancer, and more particularly mutations found in the PALB2 gene that are linked to an increased risk of cancer.
  • Fanconi anemia is a genetically heterogeneous condition with 12 complementation groups currently recognised, 11 of which have been attributed to distinct genes, FANCA (FA- A), FANCB (FA-B), FANCC (FA-C), BRCA2 (FA-Dl), FANCD2 (FA- D2), FANCE (FA-E), FANCF (FA-F), FANCG (FA-G), PALB2 (FA- J), FANCL (FA-L) and FANCM (FA-M). These genes encode interacting proteins that participate in the recognition and repair of DNA interstrand cross-links 2 .
  • BRCA2 is a DNA repair protein with a key role in the repair of double-strand DNA breaks by homologous recombination.
  • BRCA2 was originally identified through positional cloning of a familial breast cancer predisposition gene and monoallelic (heterozygous) mutations are associated with high risks of breast and ovarian cancer. Subsequently, biallelic BRCA2 mutations were found to cause a rare subtype of Fanconi anemia, FA- Dl.
  • the phenotype of biallelic BRCA2 mutations differs from other Fanconi anemia subtypes, most strikingly in the risk and spectrum of associated cancers.
  • Fanconi anemia subtypes are associated with greatly increased risks of cancer, primarily acute myelogenous leukaemia and adult squamous cell carcinomas, particularly in the oral cavity and the pharyngo-esophageal and female genital regions.
  • Childhood solid tumors occur very rarely in Fanconi anemia and, to date, have been almost exclusively associated with FA-Dl.
  • the risk of childhood embryonal tumors, particularly Wilms tumor and medulloblastoma is very high in biallelic BRCA2 mutation carriers.
  • the major known breast cancer susceptibility genes are BRCAl and BRCA2.
  • Germline mutations in BRCAl and BRCA2 confer a high relative risk of developing breast cancer, of the order of 10-20 fold by age 60.
  • the prevalence of disease-associated mutations in these genes varies in different populations, but is generally low. In the UK, approximately one in 900 individuals is heterozygous for a BRCAl mutation and one in 800 heterozygous for a BRCA2 mutation. In Ashkenazi Jews, the prevalence of mutations in both genes is higher, with approximately 1% individuals being heterozygous for mutations in each of these genes.
  • Germline mutations in the TP53 gene also confer a high risk of breast cancer. However, TP53 cancer predisposing mutations are much rarer than those in BRCAl or BRCA2.
  • CHEK2 encodes a protein kinase that modulates the activities of TP53, BRCAl and BRCA2.
  • CHEK2 mutations confer a two-fold relative risk of breast cancer and in many countries such mutations are present in 0.5-1% of the population.
  • ATM is critical in the cellular response to double stranded DNA damage and regulates the activities of BRCAl and TP53.
  • Epidemiological and molecular studies have demonstrated that inactivating mutations in ATM confer an approximately two-fold relative risk of breast cancer.
  • ATM breast cancer predisposing alleles have a similar prevalence to those in CHEK2, with 0.5-1% of the population being heterozygotes .
  • truncating mutations in BRIPl which encodes a protein that binds BRCAl and mediates some of its functions, also confer a two-fold relative risk of breast cancer.
  • BRIPl mutations appear to be rarer than those in CHEK2 and ATM, with 0.1% individuals in the UK population being heterozygotes, a prevalence similar to that of BRCAl and BRCA2 mutations in the UK.
  • Biallelic (homozygous or compound heterozygous) mutations in some breast cancer susceptibility genes cause recessive childhood syndromes characterised by sensitivity to various types of DNA damage.
  • Biallelic mutations in ATM have been recognised as the causative defects in Ataxia Telangiectasia for more than a decade.
  • biallelic mutations in BRCA2 and in BRIPl cause subgroups of Fanconi anemia (FA) , a rare, recessive, chromosomal instability disorder characterized by growth retardation, skeletal abnormalities, bone marrow failure, cancer predisposition and cellular hypersensitivity to DNA cross linking agents.
  • FA Fanconi anemia
  • the present invention is based on research to identify additional cancer susceptibility genes and in particular studies that show that mutations in PALB2 are associated with Fanconi anemia, childhood cancer, breast cancer and/or ovarian cancer.
  • PALB2 (for "partner and localizer of BRCA2") encodes a recently discovered protein that interacts with BRCA2, is implicated in its nuclear localisation and stability and is required for some functions of BRCA2 in homologous recombination and double strand .break repair.
  • PALB2 mutations, and more particularly biallelic mutations, in PALB2 are responsible for a subset of Fanconi anemia cases characterised by a high incidence of childhood solid tumors, a phenotype similar to that caused by biallelic BRCA2 mutations, we also investigated whether heterozygous (monoallelic) mutations in PALB2 confer susceptibility to breast cancer and/or ovarian cancer .
  • PALB2 (for 'partner and localizer of BRCA2') was recently identified as a nuclear partner of BRCA2 (Xia et al, 2006) .
  • PALB2 colocalises with BRCA2, promoting its localisation and stability in key nuclear structures, which in turn facilitates BRCA2 functions in DNA repair.
  • knockdown of PALB2 sensitizes cells to- MMC treatment which results in interstrand cross-links and double-strand breaks 5 .
  • Sensitivity to MMC is a hallmark of Fanconi anemia and we therefore appreciated that PALB2 might be a candidate Fanconi anemia gene.
  • the present invention represents the first proof that mutations in the PALB2 gene are linked to a susceptibility (or predisposition as the terms are used interchangeably herein) to Fanconi anemia, childhood cancer, breast cancer or ovarian cancer.
  • the association with Fanconi anemia includes cancers linked to the occurrence of this condition, including childhood embryonal tumors such as Wilms tumors, medulloblastomas, acute myelogenous leukemia (AML) and neuoblastoma .
  • the present invention provides a method of determining whether an individual has an increased susceptibility to Fanconi anemia, childhood cancer or breast cancer, the method comprising determining in a sample obtained from the individual the presence of a mutation in the PALB2 gene, or a polypeptide encoded by the PALB2 gene, wherein the presence of a mutation is indicative of the increased risk of Fanconi anemia, childhood cancer, breast cancer or ovarian cancer.
  • Examples of the mutations disclosed herein that are associated with Fanconi anemia and childhood cancer are set out in Table 1 and examples of mutations associated with breast cancer are set out in Table 2.
  • the mutations are truncating mutations.
  • the mutations associated with Fanconi anemia or ' childhood cancer are generally biallelic, while the mutations associated with breast cancer are generally monoallelic.
  • the present invention provides a method which comprises having determined whether an individual has an increased susceptibility to Fanconi anemia, childhood cancer, breast cancer or ovarian cancer according to the methods disclosed herein, one or more of the further step of:
  • the present invention provides a kit for detecting mutations in the PALB2 gene associated with a susceptibility to cancer according to any one of the preceding claims, the kit comprising:
  • the present invention provides novel nucleic acid and polypeptide sequences that includes an isolated nucleic acid molecule encoding the PALB2 gene having at least 90% nucleic acid sequence identity with the sequence as set out in SEQ ID NO: 2, wherein the nucleic acid comprises one of the mutations set out in Table 1 or Table 2.
  • the present invention further relates to a replicable vector comprising these nucleic acid sequences and to host cells transformed with the vector, e.g. for use in expressing PALB2 nucleic acid by culturing the host cells so that the polypeptide encoded by the PALB2 nucleic acid is produced.
  • the present invention also provides polypeptides encoded by these nucleic acid molecules and antibodies capable of specifically binding to the PALB2 polypeptides.
  • Tubulin or RAD50 was used as a loading control
  • Transduction of cells- from IFAR-849 with the vector pOZC-PALB2 restores PALB2 expression similar to that in a normal control, whereas the mock vector pSllEG (expressing GFP) does not.
  • the transduction leads to reduction in MMC-induced G2 phase arrest in cells transduced with pOZC-PALB2 (black) compared to that in the mock-transduced line pSllEG (grey) grown for the same time at identical MMC concentration. This effect has been reported in complemented FA cell lines from other subtypes d) FANCD2 monoubiquitination.
  • FIG. 1 Cellular phenotype of PALB2 deficiency, a) Severe chromosomal damage in a fibroblast metaphase from GESH treated with 50ng/ml MMC. b) Dose-response - curves demonstrate diminished survival of lymphocytes from patient LOAO as opposed to his unaffected sibling. The cells were transduced with eGFP or Fanconi anemia cDNAs as indicated, c) No cells with >5 RAD51 nuclear foci were detectable in fibroblasts from GESH after induction of DNA damage, whereas a normal control clearly showed nuclei
  • FIG 4. Abridged pedigrees of eight breast cancer families with PALB2 mutations. Individual screened for PALB2 mutations is indicated with an arrow. Individuals with breast cancer are shown as filled circles, with the age at diagnosis given underneath. Other cancers or medical conditions are not shown. Samples were not available from individuals with breast cancer that are not genotyped.
  • the PALB2 mutation in each family is shown in Figure 1 and Table 1 and is shown under the individual. BC, breast cancer; PALB2_WT, PALB2 mutation absent.
  • FIG. 5 Schematic diagram of the Fanconi anemia-BRCA pathway.
  • the FA-core complex consists of eight FA proteins (A, B, C, E, F, G, L and M) and is essential for the monoubiquitination and activation of FANCD2 following DNA damage.
  • Activated FANCD2 is translocated to DNA repair foci where it colocalises with other DNA damage response proteins including BRCA2 and RAD51 and participates in homology directed repair. Shaded proteins are encoded by genes that cause Fanconi anemia. Proteins outlined in blue are encoded by genes that confer susceptibility to breast cancer.
  • BRIPl, BRCA2 and PALB2 are both Fanconi anemia genes and breast cancer susceptibility genes and their encoded proteins all function downstream of FANCD2.
  • SEQ ID NO: 1 shows the amino acid sequence of PALB2.
  • SEQ ID NO: 2 shows coding sequence of the PALB2 gene. Detailed Description
  • PALB2 gene and polypeptide sequences are disclosed in Xia et al 1 , and are publicly available on GenBank as sequence accession numbers NM024675 and BC044254.
  • the polypeptide sequence is 1186 amino acids in length and is provided a SEQ ID NO: 1.
  • the coding sequence of the PALB2 gene is reproduced herein as SEQ V ID NO: 2.
  • PALB2 nucleic acid includes the sequence shown in SEQ ID NO: 2, alleles and sequence variants thereof and a complementary sequences of any of these nucleic acids.
  • the numbering used herein refers to these sequences and in particular in Tables 1 and 2 to the coding sequence of the PALB2 gene shown in SEQ ID NO: 2.
  • the present invention is also applicable to the use of alleles and sequence variants of this gene that may include one or more of the mutations as disclosed herein.
  • PALB2 nucleic acid and amino acid sequences preferably have at least 90% sequence identity, more preferably 98% sequence identity, and most preferably at least 98% sequence identity, to their respective sequences set out in SEQ ID NO: 1 and 2.
  • Percent (%) amino acid sequence identity with respect to the PALB2 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the PALB2 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the % identity values can be generated by WU-BLAST-2 which was obtained from [Altschul et al, Methods in Enzymology, 266:460-480 (1996); http://blast.wustl/edu/blast/README.html] .
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSPS and HSPS2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored) .
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the PALB2 polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the PALB2 coding sequence as provided in SEQ ID NO: 1.
  • identity values used herein were generated by the BLASTN module of WU BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • mutant alleles of the present invention are set out in Tables 1 and 2 and are described using the nomenclature Antonarakis, Recommendations for a nomenclature system for human gene mutations, Nomenclature Working Group, Hum. Mutat., 11(1): 1-3, 1998. These mutations are generally associated with the production of truncated forms of PALB2 polypeptide show in the experimental work described herein to be associated with susceptibility to cancer, and especially to Fanconi anemia, childhood cancer, breast cancer or ovarian cancer. Implications for screening, e.g. for diagnostic or prognostic purposes, are discussed below.
  • nucleic acid according to the present invention provides novel PALB2 nucleic acid sequences, in particular the mutations set out in Tables 1 and 2 or described elsewhere in the present application.
  • nucleic acid according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid sequences encoding all or part of the PALB2 gene and/or its regulatory elements can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992) . These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • the sequences can be incorporated in a vector having control sequences operably linked to the PALB2 nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the PALB2 polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • PALB2 polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the PALB2 polypeptide is produced and recovering the PALB2 polypeptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.
  • the choice of host cell can be used to control the properties of the PALB2 polypeptide expressed in those cells, e.g. controlling where the polypeptide is deposited in the host cells or affecting properties such as its glycosylation.
  • the purpose of carrying of the methods disclosed herein on a sample from an individual is to determine whether the individual carries a PALB2 allele predisposing them to cancer.
  • the purpose of such analysis may be used for diagnosis or prognosis, e.g. to serve to detect the presence of an existing cancer, to help identify the type of cancer, to assist a physician in determining the severity or likely course of the cancer and/or to optimise treatment of it.
  • the methods can be used to detect PALB2 alleles that are statistically associated with a susceptibility to cancer in the future, e.g. early onset breast cancer, identifying individuals who would benefit from regular screening to provide early diagnosis of cancer or for whom changes in lifestyle or diet may help to ameliorate the increased susceptibility to a particular form of cancer.
  • the methods divide into those screening for the presence of PALB2 nucleic acid sequences and those that rely on detecting the presence of PALB2 polypeptide. Exemplary techniques and their advantages and disadvantages are reviewed in Nature Biotechnology, 15:422-426, 1997.
  • the methods make use of biological samples from individuals that may contain the nucleic acid or polypeptides . Examples of biological samples include blood, plasma, serum, tissue samples and saliva.
  • Nucleic acid based testing may be carried out using preparations containing genomic DNA, cDNA and/or mRNA.
  • RNA is more difficult to manipulate than DNA because of the widespread occurrence of RN'ases.
  • Techniques that involve looking for mutations in PALB2 nucleic acid sequence include direct sequencing, restriction fragment length polymorphism (RFLP) analysis, single-stranded conformation polymorphism (SSCP) , PCR amplification of specific alleles, amplification of DNA target by PCR followed by a mini-sequencing assay, allelic discrimination during PCR, Genetic Bit Analysis, pyrosequencing, oligonucleotide ligation assay, or analysis of melting curves.
  • Techniques that involve looking for mutations in PALB2 polypeptides include the use of specific binding members such as antibodies to detect mutated and/or normal PALB2 polypeptides.
  • Nucleic acid in a test sample may be sequenced and the sequence compared with the sequence shown in SEQ ID NO: 1, for example to determine whether the sequence contains a truncating mutation, such as one of the mutations shown in Table 1 or Table 2, and hence is associated with a susceptibility to cancer. Since it will not generally be time or labour efficient to sequence all nucleic acid in a test sample, or even the whole PALB2 gene, a specific amplification reaction such as PCR using one or more pairs of primers may be employed to amplify the region of interest in the nucleic acid, for instance the PALB2 gene or a particular region in which mutations associated with cancer susceptibility occur. Exemplary primers for this purpose can be designed by the skilled person based on the information provided herein.
  • the amplified nucleic acid may then be sequenced as above and/or tested in any other way to determine the presence or absence of a particular feature.
  • Nucleic acid for testing may be prepared from nucleic acid removed from cells or in a library using a variety of other techniques such as restriction enzyme digest and electrophoresis.
  • Mutations in nucleic acid may also be screened using a mutant- or allele-specific probe.
  • a probe corresponds in sequence to a region of the PALB2 gene, or its complement, containing a sequence mutation known to be associated with cancer susceptibility, for example as set out in Table 1.
  • specific hybridisation of such a probe to test nucleic acid is indicative of the presence of the sequence alteration in the test nucleic acid.
  • more than one probe may be used on the same test sample. This approach may be adapted to use a microarray as discussed in more detail below.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RNAase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
  • stringent conditions include those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/ 0.1% sodium dodecyl sulfate at 50 0 C; (2) employ during hybridisation a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 760 mM sodium chloride, 75 mM sodium citrate at 42 0 C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0.1% sodium pyrophosphate, 5 x Denhardt '
  • an oligonucleotide probe will hybridise with a sequence which is not entirely complementary.
  • the conditions of the hybridisation can be controlled to minimise non-specific binding, and preferably stringent to moderately stringent hybridisation conditions are preferred.
  • the skilled person is readily able to design such probes, label them and devise suitable conditions for the hybridisation reactions, assisted by textbooks such as Sambrook et al (1989) and Ausubel et al (1992).
  • the degree of base-pairing between the two molecules will be sufficient for them to anneal despite a mismatch.
  • Various approaches are well known in the art for detecting the presence of a mismatch between two annealing nucleic acid molecules.
  • RN'ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid.
  • Other approaches rely on the use of enzymes such as resolvases or endonucleases .
  • an oligonucleotide probe that has the sequence of a region of the normal PALB2 gene (either sense or anti- sense strand) in which mutations associated with cancer susceptibility are known to occur (e.g. see Tables 1 or 2) may be annealed to test nucleic acid and the presence or absence of a mismatch determined. Detection of the presence of a mismatch may indicate the presence in the test nucleic acid of a mutation associated with cancer susceptibility.
  • an oligonucleotide probe that has the sequence of a region of the PALB2 gene including a mutation associated with cancer susceptibility may be annealed to test nucleic acid and the presence or absence of a mismatch determined. The absence of a mismatch may indicate that the nucleic acid in the test sample has the normal sequence. In either case, a plurality of probes to different regions of the gene may be employed.
  • Allele or variant-specific oligonucleotides may similarly be used in PCR to specifically amplify particular sequences if present in a test sample.
  • Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art.
  • the PCR product may for instance be treated in a way that enables one to display the mutation or polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected.
  • PCR techniques for the amplification of nucleic acid are described in US Patent No. 4683195, Mullis et al, Cold Spring Harbor Symp. Quant.
  • the presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the normal gene or a variant or allele is digested with the same enzyme or enzymes.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies), specific for one or more particular variants of the polypeptide, for example the normal PALB2 polypeptide and mutated forms thereof.
  • a binding partner for a specific binding member such as an antibody (or mixture of antibodies), specific for one or more particular variants of the polypeptide, for example the normal PALB2 polypeptide and mutated forms thereof.
  • the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding, before binding is determined, for instance using a reporter system as discussed.
  • a specific binding member such as an antibody under appropriate conditions for specific binding
  • different reporting labels may be employed for each antibody so that binding of each can be determined.
  • a specific binding member such as an antibody may be used to isolate and/or purify its binding partner polypeptide from a test sample in preference to other components that may be present in the sample. This may be used to determine whether the polypeptide has the sequence shown in SEQ ID NO: 1, or if it is a mutant form. Amino acid sequence is routine in the art using automated sequencing machines.
  • a "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • the skilled person will be able to think of many other examples and they do not need to be listed here. It has become a matter of routine in the art for the skilled person to make antibodies that are capable of specifically binding to different polypeptides.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine .
  • Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • microarrays can be particularly valuable as they can provide great sensitivity, particularly through the use of fluorescent labelled reagents, require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays can be carried out simultaneously. This latter advantage can be useful as it provides an assay for different mutations in the PALB2 gene or mutations in other genes to be carried out using a single sample, e.g. in forms of genetic profiling.
  • Microarrays are libraries of biological or chemical entities immobilised in a grid/array on a solid surface and methods for making and using microarrays are well known in the art. A variation on this theme is immobilisation of these entities onto beads, which are then formed into a grid/array.
  • the entities immobilised in the array can be referred to as probes. These probes interact with targets (a gene, mRNA, cDNA, protein, etc) and the extent of interaction is assessed using fluorescent labels, colorimetric/chromogenic labels, radioisotope labels or label-free methods (e.g. scanning Kelvin microscopy, mass spectrometry, surface plasmon resonance, etc) . The interaction may include binding, hybridization, absorption or adsorption.
  • the microarray process provides a combinatorial approach to assessing interactions between probes and targets. The basic nucleic acid microarray concept is described in US Patent No: 5700637 and 6054270.
  • a DNA microarray is a collection of microscopic DNA spots attached to a solid substrate, e.g. glass, plastic or silicon chip, forming an array.
  • DNA microarrays are now commercially available. There are three basic forms: spotted microarrays, lithographic microarrays and a bead-based systems. Each involves analysing DNA sequences by the immobilisation of cDNA probes or in situ creation of oligonucleotide sequences and subsequent hybridisation with target mRNA/cDNA complementary to the probes. Often the target cDNA are fluorescently labelled. Sequencing by hybridization approaches are described, for example, in US Patent No: 6913879, 6025136, 6018041, 5525464 and 5202231.
  • oligonucleotide sequences are built in situ base by base on the chip.
  • cDNA or oligonucleotide probes are deposited on the array using contact or non-contact printing methods .
  • oligonucleotides, cDNA or small fragments of PCR products corresponding to mRNAs are printed in an array pattern on a solid substrate by either a spotting robot using pins or variations on ink- jet printing methods.
  • the spots are typically in the 30- 500 ⁇ m size range with separations of the order of 100 ⁇ m or more.
  • a lack of uniformity of spot size, variations of spot shape and donut or ring-stain patterns caused during the drying of spots can result in non-uniform immobilisation of the DNA and hence non-uniform fluorescence following the hybridisation.
  • sequences of oligonucleotides are built up by selective protection and deprotection of localised areas of the substrate.
  • This approach has been employed, inter alia, by Affymetrix.
  • Affymetrix chips generally provide higher probe densities (spot sizes of the order of 10 ⁇ m or greater) , but have shorter sequence lengths than in spotted or bead microarrays.
  • the fluorescent labelling of target cDNA remains a key part of the detection strategy.
  • the photolithographic approach is described in US Patent No: 6045996 and 5143854.
  • An alternative method for making arrays employs bead based microarrays.
  • An example of this approach is the system used by Illumina (http://www.illumina.com/) in which probes are immobilised on small (3-5 ⁇ m diameter) beads. After hybridisation the beads are cast onto a surface and drawn into wells by surface tension. In the Illumina system, the wells are etched into the ends of optical fibres in fibre bundles. The fluorescence signal is then read for each bead. The method includes a tagging of each bead so that the bioactive agent on each bead can be decoded from the probe position and a decoding system is needed to distinguish the different probes used.
  • the bead based system is described in US Patent No: 6023540, 6327410, 6266459, 6620584 and 7033754.
  • the same principles relate to DNA microarrays, the same principles have been extended to protein and chemical microarrays.
  • the probes immobilised on the surface are specific proteins, antibodies, small molecule compounds, peptides, carbohydrates, etc rather than DNA sequences.
  • the targets are complex analytes, such as serum, total cell extracts, and whole blood. ,
  • the key concepts of an array of probes, which undergo selective binding/interaction with a target and which are then interrogated via, for example, a fluorescent signal remain central to the method.
  • the present invention provides a microarray, or the components for forming a microarray (e.g. a bead array), wherein the microarray comprises one or more binding agents present or locatable on a substrate at a plurality of locations, wherein the one or more binding agents are capable of specifically binding to PALB2 nucleic acid containing a truncating mutation or to a truncated PALB2 polypeptide encoded by the nucleic acid.
  • the microarray will preferably also comprise a plurality of further binding agents for carrying out other tests on the sample, for example to determine the presence of other mutations that are associated with a susceptibility to a disease or condition, such as cancer. Ki ts
  • kits for carrying out the methods disclosed herein The components of the kit will be dependent on whether the method is for determining the presence of a mutation in the PALB2 gene, or a polypeptide encoded by the PALB2 gene, for example the presence of a truncating mutation, or truncated polypeptide.
  • the components of the kit will be provided in a suitable form or package to protect the contents from the external environment.
  • the kit may also include instructions for its use and to assist in the interpretation of the results of the test.
  • the kit may also comprise sampling means for use in obtaining a test sample from an individual, e.g. a swab for removing cells from the buccal cavity or a syringe for removing a blood sample (such components generally being sterile).
  • the kit may comprise a microarray as described above, optionally in combination with other reagents, such as labelled developing reagents, useful for carrying out testing with the assay.
  • the microarray is preferably a nucleic acid array.
  • the kit may be for use in PCR based testing according to the methods disclosed herein and accordingly may comprise one or more primers suitable for amplifying a portion of the PALB2 nucleic acid sequence where one of the mutations associated with a susceptibility to cancer are located.
  • the kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample.
  • the kit may also one or more further reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc.
  • the nucleic acid primer may also be labelled, for example to facilitate detection and/or quantification of the amplified product.
  • the present invention provides a computer program for carrying the method for evaluating a property of a clinical treatment in a group of test subjects .
  • the present invention provides a data carrier having a program saved thereon for carrying out the method for evaluating a property of a clinical treatment in a group of test subjects.
  • the present invention provides a computer programmed to carry out the method for evaluating a property of a clinical treatment in a group of test subjects .
  • PALB2 sequencing Primers were designed to amplify the 13 exons and intron- exon boundaries of PALB2. We used a touchdown 68-50 0 C protocol to amplify all products which were sequenced using the BigDyeTerminator Cycle sequencing kit and a 3730 automated sequencer (ABI Perkin Elmer) . Sequencing traces were analysed using Mutation Surveyor software
  • PALB2 immunoblots were performed with samples containing 50 ⁇ g total protein each on 7% NuPage Tris-Acetate polyacrylamide gels (Invitrogen) . Membranes were probed with polyclonal rabbit anti-PALB2 antiserum raised against the first 120 amino acids of PALB2 at a concentration of 1:1000 (gift of B Xia) . Secondary antibody was ECL donkey anti-rabbit IgG horseradish peroxidase-linked whole antibody (GE Healthcare) at 1:2000. Detection was by the chemiluminescence technique using the ECL system (Amersham) .
  • BRCA2 immunoblots were performed with samples containing 80 ⁇ g total protein each on 3-8% NuPage Tris- Acetate polyacrylamide gels (Invitrogen). Membranes were probed with polyclonal rabbit anti-BRCA2 antiserum (Ab-2, Calbiochem) , raised against amino acids 3245-3418 of BRCA2, at a concentration of 1:200. Secondary antibody (1:5000) and detection were the same as for the PALB2 immunoblots. Immunoblotting for detection of monoubiquitinated FANCD2 was performed using previously described methods.
  • the ⁇ -retroviral vectors pOZC-PALB2 and pSllEG were packaged in PG 13 cells and used for transduction of patient and control cell lines. Gene transfer was monitored by CD25 (pOZC- PALB2) or GFP (pSllEG) expression. Transduced cells were grown for 48 h in the presence of MMC at concentrations of 12 (fibroblasts) or 15 ng/ml (LCL) . The cells were vitally stained with Hoechst 33342 fluorescent dye (Molecular Probes) at 16 ⁇ g/ml. DNA histograms were recorded by flow cytometry.
  • Lymphocyte survival was determined using CD3/CD28/IL2- stimulated lymphocytes .
  • Transduction was with FANC cDNAs (as indicated in Fig 2B) and GFP cDNA as a control, separately cloned into a Sll-type ⁇ -retroviral vector.
  • the cells were exposed to various concentrations of MMC for 5 days. Live/dead cell ratios were determined by propidium iodide exclusion/uptake on flow cytometry.
  • PALB2 predisposition in breast cancer predisposition
  • This design enhances the power of the study by exploiting the expected enrichment of predisposition alleles in familial cases and has been employed in our previous studies of CHEK2, ATM and BRIPl mutations in breast cancer susceptibility.
  • the 13 exons and intron- exon boundaries of PALB2 were PCR-amplified and sequenced in 747 breast cancer cases, each from an independently ascertained family with multiple cases of breast cancer. Each case was affected with breast cancer at any age and had at least two relatives with breast cancer. All samples were from breast cancer families ascertained through Cancer Genetics clinics in the UK.
  • a family history of Fanconi anemia was not part of the ascertainment criteria for any of the families. Families from non-UK ethnic groups were excluded. All cases had previously been screened and found negative for mutations and large deletions / duplications in BRCAl and BRCA2.
  • We quantified the extent of the family history of breast cancer in each family using a Family History Score. This was defined as the number of relatives of the index case with breast cancer, weighted by their degree of relatedness to the index case to adjust for the expected allele sharing (score l for each affected 1 st degree relatives, 0.5 for 2 nd degree relatives and 0.25 for 3 rd degree relatives; bilateral breast cancers score double) . Where possible, additional breast cancer cases from the families of index cases in whom a PALB2 mutation was found were also genotyped for that mutation. We obtained informed consent from all families and the research was approved by the London Multicentre Research Ethics Committee (MREC/01/2/18) .
  • Control samples were from the 1958 birth Cohort Collection which is an ongoing follow-up of all persons born in Great Britain during one week in 1958, including a recent biomedical assessment during 2002-2004 at which blood samples and informed consent were obtained for creation of a genetic resource. At least 97% of the controls were of white ethnicity.
  • RNA analysis in LNEY demonstrated that the second mutation in this individual, c.3350+4A>G, results in aberrant splicing and generates two abnormal products, r .3202_3350dell49, in which exon 12 is skipped, and r .3350insGCAG, which utilises a cryptic splice donor site. Both changes introduce translational frameshifts .
  • Analysis of parental DNA demonstrated that all the mutations had been inherited from different parents, consistent with autosomal recessive inheritance. No sample was available from the affected individuals LOAO, IFAR-007 and ICR-60, but their parents all carried PALB2 mutations. We also sequenced
  • PALB2 in 352 control chromosomes (176 normal individuals). No truncating or splice junction variants were identified providing further evidence that such mutations are pathogenic in the individuals with Fanconi anemia.
  • One mutation, C.3549OG, p.Y1183X was seen in two separate families.
  • a different mutation at the same nucleotide, C.3549OA also results in p.Y1183X and was identified in a third family.
  • C.3549C is in the last exon of PALB2 and there are only three amino acids after codon 1183 before the protein terminates. Truncating mutations close to the end of a protein are generally expected to escape nonsense-mediated RNA decay.
  • Fanconi anemia proteins (A, B, C, E, F, G, L and M) form a nuclear core complex that mediates monoubiquitination of FANCD2.
  • Activated FANCD2 is translocated to DNA repair foci where it colocalizes with various proteins involved in the DNA damage response, including BRCA2 (refs 6,7).
  • BRCA2 Refs 6,7.
  • PALB2 acts downstream of FANCD2 in the FA-BRCA pathway.
  • Fig ID shows normal monoubiquitination of FANCD2
  • the phenotype of FA-N is, in many ways, typical of Fanconi anemia and includes growth retardation and variable congenital malformations (Table 1) .
  • the cellular phenotype of PALB2 deficiency is similar to BRCA2 deficiency and more severe than other Fanconi anemia subtypes, with elevated spontaneous chromosome breakage rates and markedly reduced lymphocyte survival and increased chromosome breakage on exposure to MMC (Fig 2A, 2B) .
  • FA-N is associated with unusually early mortality due to childhood cancer. All seven FA-N cases developed cancer in early childhood including three Wilms tumors, five medulloblastomas, two cases of AML and one neuroblastoma (Table 1) . One individual developed three malignancies within the first year of life and three individuals had two cancers. Cancer treatment was unsuccessful in six patients, all of whom died before four years of age. LNEY is currently alive at 4.5 years but is in the early stages of treatment for medulloblastoma . The cancer spectrum of biallelic PALB2 mutations is very similar to that associated with biallelic BRCA2 mutations, which also confer high risks of embryonal tumors. The reasons for the association between childhood solid tumors and deficiency of BRCA2 or PALB2 are unclear, but seem likely to be related to functions not shared by other FA proteins.
  • Monoallelic (heterozygous) BRCA2 mutations are associated with high risks of breast and ovarian cancer and lesser risks of other cancers such as prostate and pancreatic cancer. Given the intimate functional links between PALB2 and BRCA2 and the similar phenotypes associated with biallelic mutations in the genes that encode them, it is plausible that monoallelic PALB2 mutations confer susceptibility to adult cancer. Of interest in this regard are the cancer histories in the seven FA-N families we have identified. The mother of IFAR-007 had early- onset bilateral breast cancer and has a strong family history of breast cancer affecting her sister, mother and other more distant relatives. BRCAl and BRCA2 mutations have been excluded in this family.
  • a group of further mutations in the PALB2 gene were identified as follows.
  • G796X associated with breast cancer.
  • 172delTTGT associated with breast and/or ovarian cancer.
  • W1038X - associated with breast cancer W906X - associated with breast cancer.
  • Fanconi anemia is a genetically heterogeneous recessive condition that currently includes 13 subtypes, 12 of which have been attributed to distinct genes.
  • the known FA genes encode proteins that interact in a complex, and incompletely understood, fashion to facilitate recognition and repair of DNA double-strand breaks.
  • a key process in the pathway involves eight of the known FA proteins forming a nuclear core complex that mediates monoubiquitination and activation of FANCD2.
  • Activated FANCD2 is translocated to DNA repair foci where it colocalizes with BRCA2 and other proteins that effect DNA repair by homologous recombination (Fig 5) .
  • Biallelic mutations of BRCA2 and PALB2 cause Fanconi anemia subtypes FA-Dl and FA-N respectively.
  • the phenotypes associated with biallelic BRCA2 and PALB2 mutations are strikingly similar to each other and differ from the other 10 known Fanconi anemia genes.
  • FA-Dl and FA-N are associated with high risks of solid childhood malignancies, such as Wilms tumor and medulloblastoma, which occur very rarely in other subtypes.
  • Heterozygous mutations in the BRIPl gene which encodes a BRCAl-interacting protein, also confer an elevated risk of breast cancer and biallelic BRIPl mutations cause Fanconi anemia subtype FA-J.
  • FA- J is associated with the classical Fanconi anemia phenotype and no FA-J case with a childhood solid tumor has been reported. The biological explanations for these phenotypic similarities and differences are unknown.
  • BRCAl and BRCA2 are likely to be the only major high penetrance cancer susceptibility genes. Together, mutations in these genes account for ⁇ 20% of the familial risk of breast cancer. The remaining susceptibility is therefore predominantly due to genes conferring more modest increases in risk.
  • CHEK2, ATM, BRIPl and now PALB2 have been shown to be breast cancer susceptibility genes. Compared to BRCAl and BRCA2, inactivating mutations in these four genes confer relatively small increases in risk, approximately 2-2.5 fold. The mutations conferring risk in these four genes are all relatively rare with fewer than 1% of the population being heterozygotes .

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Abstract

La présente invention concerne des matériaux et des méthodes permettant de déterminer la susceptibilité au cancer, et plus particulièrement des mutations dans le gène PALB2 qui sont liées à un risque accru d'anémie de Fanconi, de cancer infantile, de cancer du sein et de cancer de l'ovaire.
PCT/GB2007/003977 2006-10-18 2007-10-17 Matériaux et méthodes permettant de déterminer la susceptibilité au cancer Ceased WO2008047128A2 (fr)

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JP2012501651A (ja) * 2008-09-03 2012-01-26 ザ・ジョンズ・ホプキンス・ユニバーシティー 膵臓腫瘍形成の根底にある経路および遺伝性の膵癌遺伝子
EP2417268A4 (fr) * 2009-03-05 2012-07-11 Univ Johns Hopkins Procédé de diagnostic utilisant palb2
US11104956B2 (en) 2012-06-06 2021-08-31 Myriad Genetics, Inc. Hereditary cancer genes
WO2022117923A1 (fr) * 2020-12-02 2022-06-09 Nightingale Health Oyj Méthode pour déterminer si un patient présente un risque de développer une anémie et/ou un état métabolique liés à l'état nutritionnel et/ou à l'équilibre fluidique du sang
US11773449B2 (en) 2017-09-01 2023-10-03 The Hospital For Sick Children Profiling and treatment of hypermutant cancer

Family Cites Families (5)

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US5582979A (en) * 1989-04-21 1996-12-10 Marshfield Clinic Length polymorphisms in (dC-dA)n.(dG-dT)n sequences and method of using the same
US6251588B1 (en) * 1998-02-10 2001-06-26 Agilent Technologies, Inc. Method for evaluating oligonucleotide probe sequences
AU4825699A (en) * 1998-06-19 2000-01-05 Genzyme Corporation Identification and use of differentially expressed genes and polynucleotide sequences
AU5485600A (en) * 1999-06-16 2001-01-02 Johns Hopkins University, The Characterization of the yeast transcriptome
US20040029114A1 (en) * 2001-01-24 2004-02-12 Eos Technology, Inc. Methods of diagnosis of breast cancer, compositions and methods of screening for modulators of breast cancer

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JP2012501651A (ja) * 2008-09-03 2012-01-26 ザ・ジョンズ・ホプキンス・ユニバーシティー 膵臓腫瘍形成の根底にある経路および遺伝性の膵癌遺伝子
EP2326734A4 (fr) * 2008-09-03 2012-07-11 Univ Johns Hopkins Voies à l origine de la tumorigenèse pancréatique et gène héréditaire du cancer pancréatique
AU2009288097B2 (en) * 2008-09-03 2013-11-14 The Johns Hopkins University Pathways underlying pancreatic tumorigenesis and an hereditary pancreatic cancer gene
JP2015070839A (ja) * 2008-09-03 2015-04-16 ザ・ジョンズ・ホプキンス・ユニバーシティー 膵臓腫瘍形成の根底にある経路および遺伝性の膵癌遺伝子
EP2417268A4 (fr) * 2009-03-05 2012-07-11 Univ Johns Hopkins Procédé de diagnostic utilisant palb2
US9315868B2 (en) 2009-03-05 2016-04-19 The Johns Hopkins University Diagnostic method using PALB2
US11104956B2 (en) 2012-06-06 2021-08-31 Myriad Genetics, Inc. Hereditary cancer genes
US12410478B2 (en) 2012-06-06 2025-09-09 Myriad Genetics, Inc. Hereditary cancer genes
US11773449B2 (en) 2017-09-01 2023-10-03 The Hospital For Sick Children Profiling and treatment of hypermutant cancer
WO2022117923A1 (fr) * 2020-12-02 2022-06-09 Nightingale Health Oyj Méthode pour déterminer si un patient présente un risque de développer une anémie et/ou un état métabolique liés à l'état nutritionnel et/ou à l'équilibre fluidique du sang

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