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WO2008108640A1 - Gène muté dans le groupe i de complémentation de l'anémie de fanconi - Google Patents

Gène muté dans le groupe i de complémentation de l'anémie de fanconi Download PDF

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Publication number
WO2008108640A1
WO2008108640A1 PCT/NL2008/000077 NL2008000077W WO2008108640A1 WO 2008108640 A1 WO2008108640 A1 WO 2008108640A1 NL 2008000077 W NL2008000077 W NL 2008000077W WO 2008108640 A1 WO2008108640 A1 WO 2008108640A1
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Prior art keywords
fanci
gene
dna
sequence
mutation
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Josephine Christine Dorsman
Johannes Petrus De Winter
Hans Joenje
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Vereniging voor Christelijik Hoger Onderwijs Wetenschappelijk Onderzoek en Patientenzorg
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Vereniging voor Christelijik Hoger Onderwijs Wetenschappelijk Onderzoek en Patientenzorg
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 current invention relates to an isolated human genomic DNA molecule on chromosome 15 wherein said DNA molecule has a nucleotide sequence which sequence is mutated in complementation group I of Fanconi anemia (FA), the FANCI gene. It further relates to methods for determining a genetic defect in a patient, the defect being a mutation in the Fanconi anemia gene of complementation group I, or for complementing a genetic defect in an isolated cell, the defect being a mutation in the Fanconi anemia gene of complementation group I.
  • FA Fanconi anemia
  • the invention relates to the use of the gene and fragments, cDNA and fragments, polypeptides and fragments, and antibodies against FANCI and siRNAS against FANCI, for diagnosis of FA, determining hereditary predisposition, tumor classification, for treatment or for drug development.
  • Fanconi anemia is a genetically heterogeneous chromosomal instability disorder with both autosomal and X-linked recessive inheritance and characterized by developmental abnormalities, retarded growth, bone marrow failure, and a high risk of cancer (Joenje, H., & Patel, K.J. Nat. Rev. Genet. 2, 446-457 (2001); Levitus, M. et al . Cell. Oncol. 28, 3-29 (2006); Taniguchi, T. & D'Andrea, A. D. Blood 107, 4223-4233 (2006) .
  • MMC mitomycin C
  • diepoxybutane diepoxybutane
  • Thirteen complementation groups are currently distinguished, all of which - except group I - have been linked to distinct disease genes (Levitus, M. et al. Cell. Oncol. 28, 3-29 (2006); Taniguchi, T. & D'Andrea, A. D. Blood 107, 4223-4233 (2006); Xia, B. et al. Nat. Genet. 39, 159-161 (2007)).
  • FA proteins are supposed to function in the FA pathway of genomic maintenance. Most of these proteins assemble into a multiprotein core complex which functions as an E3 ubiquitin ligase to modify FANCD2 by monoubiquitination (Garcia-Higuera, I. et al. MoI. Cell 7, 249-262 (2001)). FANCJ/BRIPl, FANCD1/BRCA2, and FANCN/PALB2 are supposed to act downstream of this modification step, because FANCD2 ubiquitination appears normal in cells lacking these proteins.
  • the ubiquitinated form of FANCD2 is called FANDCD2-L, whereas the non- ubiquitinated form is called FANCD2-S.
  • FANCD2 ubiquitination Patient cell lines of complementation group I (FA-I cells) are deficient in FANCD2 ubiquitination and are characterized by a defect in the association of FANCD2 with chromatin (Levitus, M. et al . Cell. Oncol. 28, 3-29 (2006).).
  • FA-C complementation group C
  • the identification, cloning and sequencing of such a DNA molecule should facilitate new and improved methods of diagnosis and treatment of Fanconi anemia, and also cancer.
  • DNA deoxyribonucleic acid.
  • DNA is a polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA) ) .
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides, referred to as codons in DNA molecules code for amino acid in a polypeptide.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the ⁇ iRNA into which the DNA sequence is transcribed.
  • cDNA complementary DNA: a piece of DNA lacking internal, non-coding segments (see introns) and regulatory sequences which determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • FA carrier or FA heterozygote a person who does not exhibit apparent signs and symptoms of FA but whose chromosomes contain a mutant FA gene that may be transmitted to that person's offspring.
  • FA gene a gene, the mutant forms of which are associated with the disease Fanconi anemia.
  • This definition is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence do not affect the essential functions of the gene product.
  • This term relates primarily to an isolated coding sequence but can also include some or all of the flanking 5' and 3' regulatory elements and/or intron sequences, and the so-called UTR-regions (untranslated regions) .
  • FA patient a person who carries a mutant FA gene, such that the person exhibits clinical signs and/or symptoms of FA.
  • FA-I Fanconi anemia of complementation group I.
  • FA-I carrier or FA-I heterozygote a person who does not exhibit signs or symptoms of FA but whose chromosomes contain a mutant FA-I gene that may be transmitted to that person's offspring.
  • FA-I gene or FANCI the gene, present in the human genome, mutant forms of which are associated with Fanconi anemia of complementation group I.
  • This definition is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence do not affect the essential functions of the gene product.
  • This term relates primarily to an isolated coding sequence, but can also include some or all of the flanking regulatory elements and/or intron sequences.
  • FA-I cDNA a human cDNA molecule which, when transfected into FA-I cells, is able to complement the hypersensitivity of those cells to DNA crosslinking agents.
  • the FA-I cDNA is derived by reverse transcription from the mRNA encoded by the FA-I gene and lacks internal noncoding segments present in the FA-I gene.
  • FA-I protein or polypeptide the protein encoded by a human FA-I cDNA. This definition is understood to include the various sequence polymorphisms that exist, wherein amino acid substitutions in the protein sequence do not affect the essential functions of the protein.
  • Mutant FA-I gene a mutant form of the FA-I gene which is associated with Fanconi anemia of complementation group I.
  • Mutant FA-I RNA the RNA transcribed from a mutant FA-I gene.
  • Mutant FA-I protein the protein encoded by a mutant FA-I gene.
  • ORF open reading frame. Contains a series of nucleotide triplets (codons) coding for amino acids without any termination codons . These sequences are usually translatable into protein.
  • PCR polymerase chain reaction. Describes a technique in which cycles of denaturation, annealing with primer, and then extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.
  • siRNA Abbreviation for small inhibitory RNA, a short sequence of RNA which can be used to silence gene expression.
  • an isolated human DNA molecule derived from chromosome 15 wherein said DNA molecule has a nucleotide sequence which sequence is mutated in FA complementation group I and in individuals predisposed to cancer.
  • chromosome 15 comprises at least one gene that has a nucleotide sequence which sequence is mutated in FA complementation group I.
  • the isolated DNA molecule does in normal and healthy person not contain a mutation, whereas in FA complementation group I said mutation might be present.
  • said mutation found in FA complementation group I contributes to the phenotype or molecular basis of FA.
  • the DNA molecule is localized to locus 15q25-26.
  • locus 15q25-26 DNA molecules can be isolated that can be advantageously utilized to solve the problem of the current invention.
  • an isolated DNA molecule from locus 15q-26 of the human genome and said molecule does in normal and healthy person not contain a mutation, whereas in
  • said mutation found in a DNA molecule isolated from locus 15q25-26 of DNA form FA complementation group I contributes to the phenotype or molecular basis of FA.
  • the DNA molecule contains a gene, wherein the (reference) gene has 38 exons with a translation start in exon 2, and encodes a 1328 amino acid protein with 3 nuclear localization and 3 ATM/ATR phosphorylation motifs.
  • Other splice variants do exist and are contemplated.
  • ATM/ATR phosphorylation motifs are, namely phosphorylation sites for the phosphatidyl inositol 3-kinase-like kinases ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) .
  • Intron retaining mode in this case, instead of splicing out an intron, the intron is retained in the itiRNA transcript. However, the intron must be properly encoding for amino acids. The intron 's code must be properly expressible, otherwise a stop codon or a shift in the reading frame will cause the protein to be likely to be nonfunctional.
  • Exon cassette mode in this case, certain exons are spliced out to alter the sequence of amino acids in the expressed protein.
  • Said splicing variants of the gene are in particular contemplated/comprehended in the current invention.
  • DNA molecule that shows at least 80%, more preferable 90%, even more preferable 95%, most preferable 98% homology with the nucleotide sequence shown in SEQ ID No. 1, or the complementary strand of said nucleotide sequence.
  • said sequence comprising at least several introns and exons, but also including the 3' and 5' UTR (untranslated region) and being/comprising the genomic DNA molecule that contains the FANCI gene and encodes the protein, provides for the solution of the problem of the current invention.
  • said genomic gene from which for example a cDNA can be derived, is comprehended by the current invention.
  • Seq ID No 1 mutations can occur (both in exons and or introns) that might be observed in FA complementation group I and in individuals predisposed to cancer.
  • DNA molecule that shows at least 80%, more preferable 90%, even more preferable 95%, most preferable 98% homology with the nucleotide sequence shown in SEQ ID No. 1, or the complementary strand of said nucleotide sequence.
  • nucleotide sequence of the genomic gene of FANCI (Seq Id No. 1)
  • complementary DNA strands which for example can be used as basis for primers and the like, useful in the polymerase chain reaction or as hybridization probes.
  • probes and primers are particularly useful in diagnosis of FA-I carriers and sufferers.
  • the isolated human FA-I genomic gene sequence also comprehended by this invention is the (reference) cDNA derived therefrom and disclosed in SEQ ID No 2.
  • the present invention also provides for the use of the FA-I cDNA and derivatives thereof, the corresponding genomic gene and derivatives thereof and of the FA-I protein and derivatives thereof, in aspects of diagnosis and treatment of FA-I.
  • a DNA molecule according to the invention is provided, the DNA molecule being a cDNA molecule selected from the group consisting of: a. a DNA molecule having the nucleotide sequence shown in SEQ ID No. 2, or the complementary strand of said nucleotide sequence; b. A DNA molecule that shows at least 80%, more preferable 90%, even more preferable 95%, most preferable 98% homology with the nucleotide sequence shown in SEQ ID No. 2, or the complementary strand of said nucleotide sequence.
  • Said cDNA allows for efficient studying and utilization of the FANCI gene in the development of, for example, methods for diagnosis of FA, for counseling of subjects carrying FANCI mutations in cancer predisposed families, for tumor classification, as target for therapy, or as a lead for drug development.
  • said cDNA can be introduced in a vector like a plasmid and brought to expression in bacteria, yeast and or other organism. Obviously it can also be used to produce protein/polypeptides, which for example can be further used for developing antibodies, finding drugs that interact with the FA pathway, ' of in particular with the FA genes and/or proteins.
  • DNA molecule that shows at least 80%, more preferable 90%, even more preferable 95%, most preferable 98% homology with the nucleotide sequence shown in SEQ ID No. 1, or the complementary strand of said nucleotide sequence.
  • nucleotide sequence of the reference cDNA of FANCI (Seq Id No. 2)
  • complementary DNA strands which for example can be used as basis for primers and the like, useful in the polymerase chain reaction or as hybridization probes.
  • probes and primers are particularly useful in diagnosis of FA-I carriers and sufferers.
  • DNA molecules which differ in minor ways from those disclosed DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the deletion, addition or substitution of nucleotides while still encoding a protein which possesses the functional characteristic of the FANCI protein are comprehended by this invention.
  • small DNA molecules which are derived from the disclosed DNA molecules. Such small DNA molecules include oligonucleotides suitable for use as hybridization probes or polymerase chain reaction (PCR) primers.
  • the degeneracy of the genetic code further widens the scope of the present invention as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein.
  • the nucleotide sequence of the FANCI DNA could be changed at certain position to any of those codons that encode the same amino acid, without affecting the amino acid composition of the encoded protein or the characteristics of the protein.
  • variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences.
  • an oligonucleotide comprising at least 150, preferably at least 100, more preferably at least 50, even more preferably at least 20, most preferably at least 15 consecutive nucleotides of a DNA molecule according to the invention.
  • such small DNA molecules can comprise at least a segment of the genomic gene (SEQ ID 1) molecule and, for the purposes of PCR, will comprise at least a 15 nucleotide sequence and, more preferably, a 15-20 nucleotide sequence of the genomic DNA.
  • DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof.
  • a DNA molecule according to any of the previous claims, wherein a mutation has been introduced by means of insertion, deletion, and/or replacement of one or more nucleotides, and wherein said molecule encodes for a protein that, when introduced into cells from patients with Fanconi anemia of complementation group I, does not reduce the sensitivity of those cells to mitomycin C.
  • Such DNA molecule thus provides a DNA molecule that carries a mutation that contributes to the FA phenotype, in particular relates to the molecular mechanism of phenotype related to FA complementation group I. It will thus be understood by the skilled person that, for example, by random mutagenesis of DNA according to the invention and introduction thereof in cells from patients with FA, it can be easily studied whether said mutagenesis led to the formation of a DNA molecule and/or protein derived thereof that can either reduce the sensitivity to mitomycin C or not, and thus that might contain mutations that either contribute to FA or not, allowing to quickly identify such mutations. Mutations found in a manner like described above are therefore also clearly contemplated.
  • a DNA molecule according to the invention wherein a mutation has been introduced chosen from the group consisting of partial of complete exon deletion, inserted exon, protein truncation, amino acid substitution.
  • a mutation has been introduced chosen from the group consisting of partial of complete exon deletion, inserted exon, protein truncation, amino acid substitution.
  • mutations or modifications in the promoter region for example leading to silencing of the promoter and mutations in the 3' flank are also contemplated.
  • a DNA molecule wherein at least one mutation or polymorphism selected from the group consisting of mutations c.2T>C, c.670-2A>G, c.3854G>A, c.3006+3A>G, C.3853OT, c.3437_3455deletion, C.3895OT, c.l264G>C, c.3350-88A>G, C.2572OT, c.2509G>T, and c.2248T>G and polymorphism C.164OT of the nucleotide sequence as shown in SEQ ID No.2 is present. Mutation was found c.670- 2A>G in a breast cancer family negative for BRCAl and BRCA2 mutations.
  • ⁇ c.' refers to cDNA sequences
  • ⁇ +3' refers to the third nucleotide of the intron beyond an intron boundary
  • ⁇ -3' refers to a position in the intron, 3 nucleotides before the intron boundary.
  • the 670-2A>G has been found by sequencing FANCI cDNA in 90 index patients that were screened for BRCAl/BRCA2 mutations and found negative, i.e. subjects predisposed for inherited breast cancer. FANCI therefore also seems to play a role in a variety of cancers, in addition to the above-identified, such as breast cancer and ovarian cancer.
  • these mutations provide for mutants that contribute to FA in particular FA complementation group I or predispose to cancer in mono-allelic form.
  • these mutations when present in the DNA, can lead to a FA phenotype, in particular FA complementation group I lead to a FA phenotype, in particular FA complementation group I phenotype, when both alleles are affected by mutation.
  • mutants in monoallelic form of the contemplated gene can increase cancer risk.
  • mutations in monoallelic form of the contemplated gene can increase cancer risk.
  • also other mutants than can be identified as described above are contemplated.
  • a polypeptide encoded by a DNA molecule according to the invention is very useful in developing new drugs, antibodies, understanding and knowledge with respect to diagnosis, treatment and the like of FA, in particular like that is shown in- FA complementation group I patients.
  • Antibodies, both monoclonal and polyclonal, and antibody fragments, specifically recognizing FANCI protein or mutated protein as identified herein on DNA level are also encompassed by the present invention.
  • mutant polypeptides the following is noted: while the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known.
  • Amino acid substitutions are typically of single residues; insertions usually will be in the order of about from 1 to 10 amino acid residues; and deletions will usually range about from 1 to 30 residues .
  • Deletions or insertions preferably are made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • a method for determining a genetic defect in a patient the defect being a mutation in the Fanconi anemia gene of complementation group I, the method comprises determination of the sequence of the FANCI gene of said patient.
  • the invention further provides a method for determining whether a subject carries a mutant FANCI gene, comprising the steps of: a) providing a biological sample from the subject, which sample includes DNA and/or RNA, b) determining the sequence of the FANCI gene or FANCI mRNA, or a portion thereof, c) compare the determined sequence with that of SEQ ID No 1 or SEQ ID No 2.
  • the biological sample can be any suitable sample from the (human) subject, as long as it contains representative DNA or RNA of the subject that can be sequenced by techniques, know in the art, e.g. by PCR mediated sequencing.
  • the FANCI sequence can be determined by using specific oligonucleotide probes.
  • the sequenced FANCI sequence can be compared with that of wild-type, such as that of SEQ ID No 1. It is also possible to use the corresponding wild-type cDNA, e.g. as given in SEQ ID No 2. Any genetic difference is indicative for a mutation in the FANCI gene.
  • any tumor formation is heriditary; in case of the presence of a single mutated gene (heterozygous) , there is predisposition for the tumor formation.
  • step b) comprises determining the sequence of a portion of the FANCI gene encompassing one or more of the mutations as defined above, or a corresponding portion of the FANCI mRNA.
  • the invention in another embodiment, relates to a method for classification of tumors, comprising the steps of: a) providing a biological sample from the subject, which sample includes DNA and/or RNA, and/or protein, b) determining whether the FANCI gene is expressed at the RNA and/or protein level, and c) determining the sequence of the FANCI gene or FANCI mRNA, or a portion thereof, d) comparing the determined sequence with that of SEQ ID
  • FANCI gene is expressed. He can determine the presence of FANCI protein, e.g. by checking specific binding with an anti FANCI antibody, or he can determine the presence of FANCI mRNA, e.g. by RT- PCR, a well established method in the art, or he can check the methylation condition of the promoter of the FANCI gene. If the promoter is methylated, it can be concluded that the gene is not expressed. If the gene is not expressed, or to a lower extent than at normal conditions, the tumor can be classified as a FANCI defective tumor, by not expressing FANCI or to a lesser extent.
  • the next step is to establish whether the FANCI gene is mutated and to identify the said mutation(s), as explained above.
  • the sequence of the FANCI gene can be determined before, after or simultaneously with the determination of of the expression of the FANCI gene. I.e., steps b) and c) can be performed in any order, or simultaneously.
  • the classification can be made based on the mutation (s) identified.
  • the invention related to a method for drug testing, in particular for the suitability for use as an antitumorigenic drug in patients having a tumor wherein FANCI is inactive or less active, comprising the steps of: a) providing a biological sample of the patient comprising
  • DNA and/or RNA and/or protein DNA and/or RNA and/or protein, b) determining whether the FANCI gene is expressed at the RNA and/or protein level, and c) determining whether a FANCI mutation is present in the tumor DNA of the patient, d) identifying the said mutation, e) treating tumor cells from the patient ex vivo with an antitumorigenic drug, f) determine whether the antitumorigenic drug is capable of inhibiting growth of the tumor cells, g) correlating the mutation with the effectivity of the antitumorigenic drug.
  • the tumor cells from the patient can be subjected to treatment with an antitumorigenic drug, such as a polyfunctional alkylating agent, preferably a bifunctional agent, most preferably cis-platin.
  • an antitumorigenic drug such as a polyfunctional alkylating agent, preferably a bifunctional agent, most preferably cis-platin.
  • any other possible antitumorigenic drug can be used to check whether it is suitable to treat the tumor. If the drug is capable of inhibiting the growth of the tumor cells, the drug is effective for the particular tumor. So, a correlation can be made of the effectivity of the drug and the particular mutation, which is helpful for future treatments of tumors wherein the same mutations are identified. In such a case, the effectivity of one or more antitumorigenic tumors is known, and based thereon, a suitable drug therapy can be designed.
  • the tumor is preferably chosen from the group, consisting of FA, breast cancer, ovarian cancer, head-and-neck cancer, solid childhood cancer or squamous cell carcinoma.
  • the FANCI gene is possible affected, is subject of the present invention.
  • sequence shown in Seq ID No 1 contains the FANCI gene.
  • An embodiment of the present invention is thus a method for screening a subject to determine if said. subject carries a mutant
  • the method comprises the steps of: providing a biological sample obtained from the subject, which sample includes DNA or RNA, and providing an assay for detecting in the biological sample the presence of a mutant FANCI gene or a mutant FANCI mRNA.
  • This assay preferably comprises either: hybridization with oligonucleotides; PCR amplification of the FANCI gene or a part thereof using oligonucleotide primers; RT-PCR amplification of the FANCI RNA or a part thereof using oligonucleotide primers; or direct sequencing of the FANCI gene of the subjects genome using oligonucleotide primers.
  • the invention relates to a method of complementing a genetic defect in an isolated cell, the defect being a mutation in the Fanconi anemia gene of complementation group I, the method comprising introducing into the cell one or more DNA molecules according to the invention, said DNA molecules having no mutations that contribute to the FA phenotype, or more particular to the FA complementation group I phenotype, or the polypeptide derived from such DNA.
  • a DNA molecule according to the invention for methods of diagnosis, treatment or drug development, in particular for methods of diagnosis, treatment or drug development directed to the diagnosis, treatment or drug testing of FA, cancer, and bone marrow failure.
  • FANCD2-S nuclear extracts of FA-I cells suggest a function for FANCI in binding FANCD2-S to the chromatin.
  • FANCD2 As both forms of FANCD2 seem to associate with the chromatin, it is possible that FANCD2 is monoubiquitinated here.
  • the faint presence of FANCD2-L in these extracts might point to a downstream function for FANCI, in binding of FANCD2 to the chromatin, but also in the stabilization of FANCD2-L (See Levitus, M. et al. Cell. Oncol. 28, 3- 29 (2006) .) .
  • FANCA Factorin A
  • FANCB FANCB
  • FANCC FANCDl/BRCA2
  • FANCD2 FANCD2
  • FANCF FANCG
  • FANCJ/BRIPl FANCL
  • FANCM FANCN/PALB2
  • a thirteenth, FANCI has now been identified by us, which is subject of this claim.
  • crosslinking agents are preferably poly or bifunctional alkylating agents, most preferably bifunctional, such as cis- platin.
  • a high-throughput screen for small molecule inhibitors of FANCI may result in the identification of agents that specifically block FANCI. Such agents, when specifically targeted to tumor cells, may result in substantial improvement of cure rates by cross-linking agents by sensitizing otherwise non-responsive cells.
  • - Molecular diagnostics for FA patients. When new FA patients are screened the current protocol will be extended to the DNA sequencing of 1) the FANCI cDNA and the 2) the genomic FANCI sequence .
  • Tumor samples can be analysed via immunohistochemistry or Western using antibodies against
  • FANCI FANCI.
  • the levels of mRNA can be determined via techniques such as quantitative RT-PCR.
  • DNA mutations can be analysed by techniques such as DNA sequencing, DNA copy number analysis of gene or exons, LOH analysis. When these analyses point to defects, tumors may be susceptible to treatment with cross- linking reagents.
  • FANCI in human cancer cell lines, such as HeLa, increases the sensitivity towards polyfunctional alkylating agents. This feature can be exploited to identify small molecules specifically inhibiting FANCI in drug screening protocols.
  • FANCI partial defect in gene A
  • a (partial) defect in gene B function on its own is not lethal.
  • Combined defects are lethal.
  • a deliberate disruption of FANCI (gene A) in a cancer cell with a specific defect to be identified (gene B) or a deliberate disruption of a to be identified gene (gene B) in a cancer cell with a defect in FANCI (gene A) may confer synthetic lethality.
  • the identification of genes of the group B group is contemplated.
  • Example 1 Patients, cell lines, and controls.
  • DNAs were isolated from blood samples obtained from The Netherlands Blood Transfusion Service; the donors were healthy and unselected for ethnic background.
  • a genome-wide scan for genetic linkage was performed using the Applied Biosystems microsatellite polymorphism linkage mapping kit MDlO and the Weber 6B Screening set, in accordance with the manufacturer's protocols and performed with the GeneAmp PCR system 9700 (Applied Biosystems microsatellite polymorphism linkage mapping kit MDlO and the Weber 6B Screening set, in accordance with the manufacturer's protocols and performed with the GeneAmp PCR system 9700 (Applied Biosystems microsatellite polymorphism linkage mapping kit MDlO and the Weber 6B Screening set, in accordance with the manufacturer's protocols and performed with the GeneAmp PCR system 9700 (Applied
  • Genomic DNA was isolated from whole blood or lymphoblastoid cell lines from patients and family members, using a Qiagen Blood mini kit (Qiagen, Venlo, The Netherlands) .
  • the genomic DNA of patient 480 was isolated from hair follicles using a 2 h incubation with proteinase K and a Qiagen Blood mini kit. Due to the lack of sufficient DNA whole genome amplification was carried out on DNA from patients 480 and 1428, using the GenomiPhi DNA amplification kit (Amersham Biosciences, Buckinghamshire, UK) .
  • Example 3 Determination of candidate regions . The initial genome-wide genetic linkage analysis with the patients
  • EUFA592 and BD952 from consanguineous families 1 and 2 ( Figure 1) and the multiplex family 4 yielded candidate regions on chromosome 2, 4, 6, 7, 8, 15, 16, 17 and 18, which were further analyzed using patient 1428 from family 2, patient EUFA1355 from family 4, and family 3.
  • the kinase DBF4/ASK (on chromosome 7q21.3) for its role in replication initiation and S-phase progression) ; the putative E2 ubiquitin conjugating enzyme FLJlIOIl (on chromosome 8q21.11 for its interaction with FANCD2 in Drosophila (FlyGrid) ; the aprataxin like HIT domain containing hydrolase LOC390637 (on chromosome 15q26.1) for its putative role in DNA repair; the RING finger Nsel (on chromosome 16pl2.1), for its role in DNA damage response as part of the SMC5/6 complex; the RING finger RNF40 (on chromosome 16pll.2), for its putative function as E3 ubiquitin ligase; and the vitamin K epoxide reductase complex subunit 1 (VKORCl on chromosome 16pll.2), for its presence in a cDNA expression
  • NP_859058.1, NP 001013679.2, NP 073581.1, XP 933746.1, and XP 934096 were subjected to a WoLFPSort and NUCDISC search (wolfpsort.org) and those were selected for which the nucleus was the most likely location and which contained at least 1 putative nuclear localization signal (NLS: pat4, pat7 or bipartite): KIAA1794 [NP_060663] ,
  • KIAA1794 is an orphan protein displaying a similar conservation as FANCD2 (human versus mouse: ⁇ 75%; both genes are present in Drosophila) . It showed an expected expression pattern for a FA gene (low and ubiquitous, but relatively prevalent in bone marrow and thymus; same pattern also found for FANCM) and contained 3 ATM/ATR motifs. C15orf42 was less conserved in the mouse than KIAA1794 (68% versus 75%) , displayed a higher level of expression than usually found for FA genes and contained 1 ATM/ATR motif. Thus in total, KIAA1794 was considered the prime candidate.
  • CTTTTTGGAAGTTTGTGGCG cFANCI.forOl TTTTTCGTAGCCAGGGCAG cFANCI.revOl CCATTTTCCAGGACCATTATTG cFANCI.for02 GGCACAGTGACAACATCCAATAG cFANCI.revO2 TCAAG ⁇ AATACC ⁇ CCTTTGGTCTATC cFANCI.for03 TTCTTCACTACTTCCAAGATCATGG cFANCI.revO3 TCTGTCTGTAACAAGAATACAAAGATTTC cFANCI.for04 GAAGAACAACTTTGAAGAACTAAGGG cFANCI.revO4 CGGAGCTA ⁇ TATCCTGTTGGAA ⁇ C • cFANCI.for05 GGCAAAAAGTTTCATTGGCG cFANCI.revO5 GGGTTTTTGCTGCTCCTGAAG cFANCI.forO ⁇ GGTCTTCGTAGAATGCCTCTTCC cFANCI.revO
  • PCR were performed under conditions known to the skilled person, using standard methodology.
  • PCR products were purified using a SAP/EXO treatment (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Sequencing reactions were prepared using specific primers and Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) . Samples were analyzed on an ABI 3730 DNA Analyzer (Applied Biosystems) .
  • Example 7 A gene mutated in Fanconi anemia complementation group I Using the methods described in the above-mentioned samples, a genome- wide linkage study involving 4 genetically informative families, including two first cousin marriages (See Fig. 1 and Table 1) , resulted in 4 candidate regions that were considered to harbour the gene: on chromosome 7q between markers D7S2204 and D7S820 (5.6 Mb, 8.6 cM, 12 genes), on 15q between D15S653 and D15S652 (7.1 Mb, 10.5 cM, 79 genes), on 16q between VKORCl and D16S3105 (14.4 Mb, 1.5 cM, 102 genes), and on 17q between D17S1290 and D17S2059 (12.3 Mb, 15.3 cM, 158 genes), together encompassing 39.4 Mb of genomic DNA and 351 genes .
  • KIAA1794 which is localized to 15q25-26, has 38 exons with a translation start in exon 2, encoding a 1328 amino acid protein with 3 nuclear localization and 3 ATM/ATR phosphorylation motifs (see Seq Id No 2) .
  • Patient BD952 was homozygous for two missense mutations, C.164OT (in exon 4) and c.3854G>A (in exon 36), resulting in a Proline to Leucine substitution at position 55 and an Arginine to Glutamine substitution at position 1285, respectively (Table 2) .
  • C.164OT in exon 4
  • c.3854G>A in exon 36
  • the maternal allele contained a premature stop (c.3853OT) in exon 37, whereas the paternal allele carried a mutation (c.3350-88A>G) in intron 31 resulting in aberrant splicing. From this individual a lymphoblastoid subline had been obtained that was phenotypically reverted to MMC resistance, while these cells had regained their capacity to monoubiquitinate FANCD2 (Fig. 2a and b) .
  • FANCI A striking feature of FA-I cells is their apparent deficiency in the association of FANCD2 with chromatin.
  • FANCI possesses several strong SQD/SQE motifs for ATM- or ATR-induced phosphorylation in its C- terminal domain, a feature that suggests a role in a DNA damage response.
  • the splice site mutation in patient EUFA695 results in an in-frame deletion of exon 27, encoding one of the SQE motifs, while the missense mutation in BD952 creates an additional SQD motif.
  • FANCI could thus be a signal-regulated localizer of FANCD2.

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Abstract

La présente invention concerne une molécule d'ADN humain isolée sur le chromosome 15. Ladite molécule d'ADN présente une séquence nucléotide qui est mutée dans les deux allèles d'individus de groupe I de complémentation de l'anémie de Fanconi (AF), le gène FANCI. L'invention concerne également des mutations monoalléliques chez des individus prédisposés au cancer. L'invention concerne en outre l'ADNc, le fragment, ainsi que les polypeptides et l'ARNsi qui en sont dérivés. L'invention porte également sur des procédés permettant de déterminer un défaut génétique chez un patient, ledit défaut étant une mutation du gène de l'anémie de Fanconi du groupe I de complémentation, ou des procédés permettant de complémenter un défaut génétique dans une cellule isolée, ledit défaut étant une mutation dans le gène de l'anémie de Fanconi du groupe I de complémentation. L'invention décrit par ailleurs la détermination des défauts chez des individus prédisposés au cancer. Enfin, l'invention concerne l'utilisation du gène, de l'ADNc, des polypeptides, ainsi que de l'ARNsi et des fragments correspondant dans le diagnostic de cancers (communs) sporadiques, ainsi que le traitement de ces cancers ou la mise au point de médicaments.
PCT/NL2008/000077 2007-03-07 2008-03-07 Gène muté dans le groupe i de complémentation de l'anémie de fanconi Ceased WO2008108640A1 (fr)

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US20130280258A1 (en) * 2007-03-12 2013-10-24 Miltenyi Biotec Gmbh Prognostic, diagnostic, and cancer therapeutic uses of fanci and fanci modulating agents

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DATABASE EMBL [online] 11 June 2002 (2002-06-11), "Homo sapiens chromosome 15, clone RP11-217B1, complete sequence.", XP002486637, retrieved from EBI accession no. EMBL:AC124068 Database accession no. AC124068 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130280258A1 (en) * 2007-03-12 2013-10-24 Miltenyi Biotec Gmbh Prognostic, diagnostic, and cancer therapeutic uses of fanci and fanci modulating agents

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