Classifications

• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to the use of probes relating to the FANCC, DAD1 GRIM19 and HADHII genes for the characterization of a biological sample.
• the invention also relates to the use of these probes for the identification of a compound intended for the treatment of psoriasis, a method of identification as well as a new research tool.
• Psoriasis is a frequent dermatological pathology which affects 3 to 5% of the European population. Psoriasis is commonly associated with other pathologies, such as hypertension, arthritis, or AIDS (Farber EM, Nall L, In Roenigk HH, Maibach Hl (Eds.): Psoriasis Third Edition, Revised and Expanded (1998), chapter 9: 107-157; Duvic M., J Invest Dermatol (1990 Nov); 95 (5): 38S-40S).
• psoriasis Based on the age of onset of symptoms and the association with certain genes of the major histocompatibility complex type I (MHC I), psoriasis has been classified into two subtypes called I and II (Henseler T, Christophers E, In Roenigk HH, Maibach Hl (Eds.): Psoriasis Third Edition, Revised and Expanded (1998), chapter 10: 159-166). Studies have also shown that 30 to 50% of psoriasis patients have a family history with one of the parents of 1 or 2 nd degree. These observations have made it possible to suggest that psoriasis is a genetic disease with an autoimmune component (Bos, Br. J. Dermatol. (1988) 118: 141; Baadsgaard et al., J. Invest. Dermatol. (1990) 95: 328 ).
• the main pathophysiological components of psoriasis include hyperproliferation associated with abnormal differentiation of keratinocytes of the epidermis, an inflammation characterized by an inflammatory infiltrate (mainly activated helper T lymphocytes) in the dermis and epidermis and by the release of pro-inflammatory cytokines, and finally a abnormal vascularization of the dermis (Krueger JG, J Am Acad Dermatol (2002 Jan); 46 (1): 1-23; quiz 23-6).
• inflammatory infiltrate mainly activated helper T lymphocytes
• psoriasis The involvement of the inflammatory process in the pathophysiology of psoriasis is supported by the mechanism of action of certain agents having a therapeutic effect beneficial for the disease.
• cyclosporine used in the treatment of severe forms of psoriasis, exerts an inhibitory effect on the synthesis of interleukin-2, a cytokine promoting the proliferation of T lymphocytes and the secretion of inflammatory cytokines by these cells. Consequently, the modulators of inflammation make it possible to limit the inflammatory component characterizing in part psoriasis.
• psoriasis is defined by an hyperplasia of the epidermis, as well as by scaling, erythema and inflammation of the skin.
• Apoptosis is a phenomenon of cell death (described among others by KERR J.F.R. et al., J. Cancer (1972), 265, 239) which is a highly selective form of cell suicide. It is characterized by easily observable morphological and biochemical phenomena. Thus, we observe a condensation of the chromatin associated or not with an endonuclease activity, the formation of apoptotic bodies and a fragmentation of deoxyribonucleic acid (DNA) due to the activation of endonucleases, in DNA fragments of 180 -200 base pairs giving a profile easily recognizable by electrophoresis on agarose gel. Apoptosis is involved in the development, differentiation and tissue renewal.
• cDNA filters arrays
• arrays are one of the technologies allowing to analyze simultaneously and quickly the expression of several hundred mRNAs from a skin biopsy taken from the patient, as opposed to other heavier technologies to implement, such as SAGE (“Sériai Analysis of Gène Expression”) (Velculescu VE, Zhang L, Vogelstein B, Kinzler KW , Science (1995 Oct) 20; 270 (5235): 484-7), or the Differential Display (Liang P, Pardee AB, Science (1992); 257: 967-971; Zhang L, et al., Science (1997 ); 276: 1268-1272).
• SAGE Supplementai Analysis of Gène Expression
• Velculescu VE Zhang L, Vogelstein B, Kinzler KW , Science (1995 Oct) 20; 270 (5235): 484-7
• Differential Display Liang P, Pardee AB, Science (1992); 257: 967-971; Zhang L, et al., Science (1997 ); 276: 12
• the Applicant has identified new marker genes for psoriasis.
• the present invention relates to probes relating to the FANCC, DAD1, GRIM19 and HADHII genes for the characterization of a biological sample, and also a new tool for characterizing a compound intended for the treatment of psoriasis, as well as a new tool of research.
• the invention relates to the use of at least one polynucleotide probe relating, respectively, to the FANCC, DAD1, GRIM19 and HADHII gene having a sequence of 10 to 500 base pairs and being complementary to at least 80% with a target sequence corresponding to all or part, respectively, of the FANCC gene (accession number NM 000136.1), DAD1 (accession number NM 001344.1), GRIM19 (accession number NM 015965.3), or HADHII ( Accession number NM 004493.1) as a psoriasis research tool.
• At least one polynucleotide probe according to the invention allows in particular the characterization of a biological sample by measuring the level of expression of one or more genes chosen from FANCC, DAD1, GRIM19 and HADHII.
• a biological sample corresponds to any sample or sample of living organism, preferably a mammal, in particular a human organism, in an amount sufficient to be characterized. Mention may be made, as a biological sample, by way of nonlimiting example, of a sample of skin, scalp, mucous membrane, or cell.
• the characterization of a biological sample consists in measuring the level of expression of a gene of interest, that is to say more precisely the quantity of messenger RNA or protein corresponding to this gene which is present in the biological sample.
• polynucleotide probe is meant a single or double-stranded oligo- or polynucleotide, of RNA or DNA, the sequence of which is complementary to a target sequence corresponding to part or all of said gene and which can range from 10 to conventionally 500 base pairs and whose sequence is complementary to the target sequence by at least 80% and preferably by at least 90% (percentage of nucleic acid bases paired between two sequences, see the calculation methods proposed by Atschul et al J. Molec. Biol. (1990), 215: 403).
• the polynucleotide probes may also have a modified backbone giving it, for example, better stability.
• the phosphodiester bonds of the natural RNA strands can be modified to include at least one nitrogen or sulfur atom.
• the polynucleotide probes can be modified so that their phosphodiester bonds are replaced by peptide-like sequences (peptide nucleic acids).
• polynucleotide probes according to the invention can comprise bases other than the 4 conventional bases.
• the polynucleotide probes according to the invention can be synthesized according to numerous synthesis methods in vivo or in vitro manually or automatically.
• RNA polymerase for example T3, T7 or SP6 RNA polymerase
• a single-stranded cDNA probe can be generated by reverse transcription of an mRNA of interest in the presence of a reverse transcriptase (for example M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase isolated from Escherichia coli).
• a reverse transcriptase for example M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase isolated from Escherichia coli.
• double-stranded RNA can then be denatured to serve as a single stranded polynucleotide.
• the polynucleotide probe can be or be part of a plasmid, a viral genome or another type of vector. It can, in other cases, be part of the genome of a cell, or else of a cell genetically modified to contain this probe in its genome. It can also be an isolated molecule.
• this probe is preferably under the control of regulatory sequences. If the probe is inserted into a vector, said vector preferably comprises all the sequences necessary for the transcription and optionally the translation of the probe. This probe can also be surrounded by flanking regions allowing a step of homologous recombination with another polynucleotide fragment, possibly leading to the insertion of the probe of the invention into the genomic DNA of a target cell.
• the invention also relates to the use of at least one polypeptide probe relating to the expression product of a gene chosen from FANCC, DAD1, GRIM19 and HADHII as a tool for researching psoriasis.
• At least one polypeptide probe according to the invention makes it possible in particular to characterize a biological sample by measuring the level of expression of the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene.
• polypeptide probe relating to the expression product of the FANCC, DAD1, GRIM19 or HADHII gene is meant a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression, respectively, of the FANCC, DAD1, GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein.
• Said antibody can be obtained according to methods known to those skilled in the art (Catty D (Ed): Antibodies, A Practical Approach, Volume I (1989), chapters 2, 3 and 4; or Harlow E. and Lane D. Antibodies, A laboratory Manual (1988), chapters 6 and 7.) using said wild protein or said homologous polypeptide.
• antibodies may, for example, be mono- or polyclonal antibodies, or their biologically active fragment capable of recognizing the protein encoded by the gene of interest.
• These antibodies can be labeled, for example immuno-conjugated with enzymes or labeled using fluorescent compounds, biotin or even radiolabelled.
• homologous polypeptides polypeptides whose amino acid sequences have at least 80%, and preferably 90% amino acids in common with the wild protein encoded by the gene of interest.
• variant polypeptide is intended to denote a polypeptide mutated or corresponding to a polymorphism which may exist, in particular in humans and which may have a truncation, a substitution, a deletion and / or an addition of at least one amino acid relative to the sequence of the normal wild-type polypeptide.
• modified polypeptide is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis, exhibiting a modification with respect to the normal wild-type sequence.
• modifications may in particular relate to the pre-, pro- or mature domains of the polypeptides, to the amino acids at the origin of a spectrum specificity or of activity efficiency, or at the origin of the structural conformation. , charge or hydrophobicity, and the capacity for multimerization and membrane insertion of the polypeptides. It will thus be possible to create polypeptides of equivalent, narrower or wider activity.
• the modifications may also relate to the sequences involved in the processing, transport and addressing of the polypeptides.
• biologically active fragment of a polypeptide is intended to denote a polypeptide fragment having conserved at least one activity of the polypeptide from which it is derived.
• FANCC Fluorescence Activated neutrophilic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic
• FANCC regulates cell apoptosis induced by gamma interferon (Pang Q, et al. EMBO J (2001 Aug) 15; 20 (16): 4478-89; Rathbun RK et al. Blood (2000 Dec) 15; 96 ( 13): 4204-11 Comment in: Blood. (2002 Apr) 1; 99 (7): 2627-8; discussion 2629-30) and TNF alpha (Pang Q, et al. EMBO J (2001 Aug) 15; 20 (16): 4478-89), which are two cytokines whose expression is increased in psoriasis (Chodorowska G.
• FANCC also regulates apoptosis induced by oxidative stress or certain xenobiotics by modulating the activity of gluthatione-S-transferase P1 (GSTP1), responsible for the detoxification of these pro-apoptotic agents (Cumming RC et al. Nat Med ( 2001 Jul) 7 (7): 814-20 Comment in: Nat Med. (2001 Dec) 7 (12): 1259-60; Pagano G.
• oxidative stress has been described as one of the potential etiological factors of psoriasis with in particular an increase in antioxidant activity in the disease (Shilov VN, Sergienko VI. Bull Exp Biol Med (2000 Apr) 129 (4): 309-13). FANCC could therefore play a role in the mechanism involving oxidative stress in the disease.
• FANCC plays a role in cell proliferation (Nadler JJ, Braun RE. Genesis (2000 Jul) 27 (3): 117-23; Haneline LS, Gobbett TA, Ramani R et al. Blood (1999 Jul) 1; 94 (1): 1-8).
• DAD1 plays a role in apoptosis (Hong NA et al. Dev Biol (2000 Apr) 1; 220 (1): 76-84; Nakashima T et al. Mol Cell Biol (1993 Oct) 13 (10): 6367- 74), and modulates the proliferation of T lymphocytes (Hong NA et al. J Immunol (1999 Aug) 15; 163 (4): 1888-93), which are important cells in the pathophysiology of psoriasis (Krueger JG, J Am Acad Dermatol (2002 Jan) 46 (1): 1-23; quiz 23-6).
• GRIM19 is a modulator of apoptosis induced by interferon-beta and retinoic acid (Angell JE, Lindner DJ, Shapiro PS et al. J Biol Chem (2000 Oct) 27; 275 (43): 33416-26) .
• HADHII hydroxyacyl-CoA dehydrogenase type II
• SDR short-chain dehydrogenases / reductases
• HADHII is also involved in apoptosis and cell proliferation. Indeed, HADHII binds the amyloid peptide A-beta, which exerts regulatory effects on apoptosis. In addition, the amyloid A-beta seems to modulate cell proliferation, since its expression is associated with a senescent state of the cells (Adler MJ., Coronel C, Shelton E. Proc Natl Acad Sci USA (1991 Jan) 1; 88 ( 1): 16-20).
• the present invention relates to the use of at least one polynucleotide probe relating to the FANCC, DAD1, GRIM19 or HADHII gene as defined above for diagnosing psoriasis or a predisposition to psoriasis by measuring the level of d expression, respectively, of the FANCC, DAD1, GRIM19 or HADHII gene.
• the present invention also relates to the use of at least one polynucleotide probe relating to the FANCC, DAD1, GRIM19 or HADHII gene as defined above for the identification of a compound intended for the treatment of psoriasis.
• the polynucleotide probe relating to the FANCC gene is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the simple cDNA probe strand of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.
• the polynucleotide probe relating to the DAD1 gene is a fragment of 10 to 500 pairs of bases representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 6 or with the double strand cDNA probe of sequence SEQ. ID. n ° 7 or with the RNA probe of sequence SEQ. ID. # 8.
• the polynucleotide probe relating to the GRIM19 gene is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the simple cDNA probe strand of sequence SEQD. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the RNA probe of sequence SEQ. ID. # 12.
• the relative polynucleotide probe having the HADHII gene is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the simple cDNA probe strand of SED sequence. ID. n ° 14 or with the double strand cDNA probe of sequence SEQ. ID. n ° 15 or with the RNA probe of sequence SEQ. ID. # 16.
• polynucleotide probe according to the invention will be used in the method described below.
• the invention thus relates to a method of identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the quantity of messenger RNA corresponding to the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene in the biological samples using at least one polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part, respectively, of the FANCC gene (no.
• GRIM19 and / or HADHII or the expression of the corresponding proteins is measured in the sample treated in step b).
• Modulation of the transcription of a gene or the expression of the corresponding proteins is understood to mean an increase or a decrease in the quantity of messenger RNA or of the protein corresponding to said gene in a test sample.
• step c) of the method can be implemented with: - a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, d homology with the single-strand cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4;
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 6 or with the double strand cDNA probe of sequence SEQ. ID. n ° 7 or with the sequence RNA probe
• a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the RNA probe of sequence SEQ. ID. # 12; a relative polynucleotide probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 14 or with the double strand cDNA probe of sequence SEQ. ID. # 15 or with the RNA sequence probe
• the measurement of the expression of the gene (s) FANCC and / or DAD1 and / or GRIM19 and / or HADHII in a biological sample can be carried out using the technology of cDNA filters ("Arrays") on nylon support, according to which the mRNAs extracted from the biological sample are transcribed into radiolabelled single-stranded cDNAs which are then deposited on cDNA fliters.
• Arrays cDNA filters
• the total mRNAs extracted from the biological sample tested can be analyzed according to one of the detection methods known to those skilled in the art (Sambrook et al.: Molecular Cloning, A Laboratory Manual , Volume I (1989) Second Edition, chapter 7) as the technique of hybridization by Northern-Blot using a denatured double stranded cDNA probe.
• the labeling of the probe may comprise a cycle of synthesis of double-stranded cDNA, from the total mRNAs extracted from the biological sample, coupled to an in vitro transcription in the presence of biotinylated ribonucleotides, generating Complementary labeled RNAs, these labeled antisense RNAs serving as a probe during hybridization on a DNA chip (Affymetrix, Gene Expression Monitoring, Technical Note; US 5,716,785, US 5,891,636, US 6,291,170).
• the present invention relates to the use of at least one polypeptide probe relating to the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene as defined above for diagnosing psoriasis or a predisposition to psoriasis of a sample biological by measuring the level of expression, respectively, of the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene.
• the present invention also relates to the use of at least one polypeptide probe relating to the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene as defined above for the identification of a compound intended for the treatment psoriasis.
• the present invention also relates to a method for identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the quantity of protein produced by the expression of at least one gene chosen from FANCC and / or DAD1 and / or GRIM19 and / or HADHII in biological samples using at least one polypeptide probe , said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression, respectively, of the FANCC, DAD1, GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or also against a homologous polypeptide, modified or variant of wild protein; d) selection of said compounds for which a modulation of the expression of said protein or proteins is measured in the sample treated in step b).
• the present invention relates to a research tool comprising a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the FANCC gene (accession number NM 000136.1) or DAD1 (accession number NM 001344.1) or GRIM19 (accession number NM 015965.3) or HADHII (accession number NM 004493.1).
• the polynucleotide probe is:
• a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4;
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 6 or with the double strand cDNA probe of sequence SEQ. ID. n ° 7 or with the RNA probe of sequence SEQ. ID. # 8;
• a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the RNA probe of sequence SEQ. ID. # 12; a relative polynucleotide probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 14 or with the double strand cDNA probe of sequence SEQ. ID. n ° 15 or with the RNA probe of sequence SEQ. ID. # 16.
• the present invention also relates to a research tool comprising a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the FANCC or DAD1 or GRIM19 or HADHII gene, or against a biologically fragment active of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein.
• the research tool may also allow the analysis of the effects of certain treatments on the expression of these marker genes for psoriasis, such as FANCC, DAD1, GRIM19 and HADHII, or the mutation analysis of the sequence of these genes in individuals with psoriasis.
• the research tool may also allow the analysis of the expression profile of the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene in the cells constituting the epidermis, in particular the keratinocytes, for example according to their state of proliferation or differentiation.
• the search tool may also allow constitutive expression, overexpression or inhibition of expression of the FANCC and / or DAD1 and / or GRIM19 and / or HADHII gene, according to in vitro or in vivo tests known to the skilled in the art.
• the polynucleotide probe according to the invention can be used, for example in a hybridization test by
• FANCC FANCC, DAD1, GRIM19 and HADHII or of a homologous, variant, modified or partial sequence, in cells constituting the epidermis, in particular keratinocytes, expressing in vitro at least one gene chosen from FANCC, DAD1,
• GRIM19 and HADHII for example depending on the state of differentiation or proliferation of cells.
• said expression can be constitutive or even inducible, the FANCC, DAD1, GRIM19 or HADHII gene being able to be placed under the control of a constitutive promoter such as the SV40 or CMV promoter, or of an inducible promoter such as the system Tet Off Gene Expression
• the polynucleotide probe according to the invention can be used, for example in a hybridization test by Northern blot, or even in an amplification test by PCR (“Polymerase Chain Reaction”), to follow the expression of the FANCC or DAD1 or GRIM19 or HADHII gene or of a homologous, variant, modified or partial, in the cells constituting the epidermis of a strain of laboratory animals, in particular in keratinocytes, overexpressing said gene or sequence in vivo, for example under the control of a promoter specifically activated in the epidermis, such as the promoters of Keratin K14, K15 or Involucrine, for example depending on the state of differentiation or proliferation of the cells.
• a promoter specifically activated in the epidermis such as the promoters of Keratin K14, K15 or Involucrine, for example depending on the state of differentiation or proliferation of the cells.
• the polynucleotide probe according to the invention can be used for the analysis, for example by Southern blotting, of an inactivation by deletion of the FANCC or DAD1 or GRIM19 or HADHII gene, for example in constituent cells.
• epidermis in particular keratinocytes, cultured in vitro or even in vivo in the cells of a strain of laboratory animals.
• This inactivation can be carried out according to conventional techniques of homologous recombination.
• the polynucleotide probe according to the invention can be used to draw and synthesize siRNAs, according to techniques known to those skilled in the art (Tuschl T, Borkhardt A, Mol Interv (2002) Jun ; 2 (3): 158-67).
• These antisense RNAs can be used for the inhibition of the expression of the FANCC or DAD1 or GRIM19 or HADHII gene, for example in a line of constituent cells of the epidermis, in particular keratinocytes, in vitro, or even in vivo, for example in a strain of laboratory animals.
• the research tool may also allow analysis of the effects of certain treatments on the expression of these psoriasis marker genes, or analysis of the mutation in the sequence of these genes in individuals suffering from psoriasis.
• this search tool could be such that the probe or probes that it comprises, are brought into contact with the corresponding target sequence (s) deposited on a support such as a filter in nylon.
• the probe can also be used in a hybridization test (for example a mismatch detection test), sequencing, microsequencing.
• the probe according to the invention may be associated with another detectable probe, the nature of which may preferably be fluorescent, radioactive or enzymatic.
• This characteristic can make it possible, among other things, to track the location of the probe, from the extracellular medium to the cell, or from the cytoplasm to the nucleus, or else to specify its interaction with DNA, or RNA or proteins.
• detectable probe is best suited as a function of the characteristic that they want to be able to follow.
• the polypeptide probe according to the invention can be used for immunolabelling of the target polypeptide as defined above, on tissue, according to conventional techniques known to those skilled in the art, for example on section of epidermis of laboratory animals, for example animals exhibiting an overexpression of the FANCC or DAD1 or GRIM19 or HADHII protein or of a homologous, variant, modified or active fragment of said protein, or even in vitro on cells constituting the epidermis, in particular keratinocytes, which may be the subject of an overexpression of the protein or of said polypeptide, or else of an inhibition of this expression, for example by the use of siRNA as envisaged previously.
• the polypeptide probe can also be used to purify said protein or said polypeptide, for example according to immunoprecipitation techniques conventionally known to those skilled in the art.
• the present invention provides a method for characterizing skin samples to determine predisposition to psoriasis.
• the Applicant has demonstrated that the expression of the FANCC, DAD1, GRIM19 and HADHII genes is induced in psoriatic lesional skin with a plaque of psoriasis, compared to healthy skin (average expression measured in 13 psoriatic subjects and 10 healthy subjects). The results obtained are presented in Table 1.
• Measuring the expression of one of the genes, and preferably the four, FANCC, DAD1, GRIM19 and HADHII makes it possible to characterize lesional psoriatic skin independently of the type and location of the psoriasis plaque (Tables 2 and 3 ).
• Table 2 shows the expression of the FANCC, DAD1, GRIM19 and HADHII genes relative to an average expression in healthy subjects, in patients with predominantly trunk and asymmetric plaques.
• Table 3 represents the expression of the FANCC, DAD1, GRIM19 and HADHII genes relative to an average expression in healthy subjects, in patients presenting mainly symmetrical plaques.
• the invention also relates to a method for diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of at least one of the FANCC, DAD1, GRIM19 or HADHII genes by putting in contact with at least one polynucleotide probe with the target sequences isolated from said biological sample, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the FANCC gene (NM accession number 000136.1) or DAD1 (NM accession number 001344.1) or GRIM19 (NM accession number 015965.3) or HADHII (NM accession number 004493.1).
• polynucleotide probe is:
• a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4;
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. # 6 or with the double cDNA probe strand of sequence SEQ. ID. n ° 7 or with the RNA probe of sequence SEQ. ID. # 8;
• a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the RNA probe of sequence SEQ. ID. # 12;
• a relative polynucleotide probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 14 or with the double strand cDNA probe of sequence SEQ. ID. n ° 15 or with the RNA probe of sequence SEQ. ID. # 16.
• the invention also relates to a method for diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of the protein produced by the expression of the FANCC or DAD1 or GRIM19 or HADHII gene by contacting a polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression, respectively, of the FANCC or DAD1 or GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein.
• a polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression, respectively, of the FANCC or DAD1 or GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide
• overexpression of a gene in a biological sample is meant a higher expression of the messenger RNA or of the protein corresponding to this gene in this sample compared to a healthy reference sample.
• the invention also relates to a diagnostic kit comprising at least one polynucleotide or polypeptide probe relating to at least one of the FANCC, DAD1, GRIM19 and HADHII genes.
• the object of the present invention therefore relates to a diagnostic kit comprising at least one polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the FANCC gene (accession number NM 000136.1) and / or DAD1 (accession number NM 001344.1) and / or GRIM19 (accession number NM 015965.3) and / or HADHII (accession number NM 004493.1).
• said polynucleotide probe of the diagnostic kit is:
• a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the sequence RNA probe
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 6 or with the double strand cDNA probe of sequence SEQ. ID. n ° 7 or with the RNA probe of sequence SEQ. ID. # 8;
• a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the RNA probe of sequence SEQ. ID. # 12;
• a relative polynucleotide probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. # 14 or with the double cDNA probe strand of sequence SEQ. ID. n ° 15 or with the RNA probe of sequence SEQ. ID. # 16.
• the subject of the present invention also relates to a diagnostic kit comprising at least one polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the FANCC or DAD1 or GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein.
• a polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the FANCC or DAD1 or GRIM19 or HADHII gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein.
• FANCC FANCC
• DAD1 DAD1
• GRIM19 HADHII
• Another object of the present invention relates to the use of a polynucleotide probe or one of its analogues, complementary, respectively, to the FANCC or DAD1 or GRIM19 or HADHII gene to modulate, respectively, the expression of the FANCC or DAD1 or GRIM19 or HADHII gene.
• the polynucleotide probe may be an antisense RNA or a siRNA which has the role of inhibiting the expression of a specific gene (Fire A., Trends in Genetic (1999) 15: 358-363).
• the polynucleotide probe is:
• a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the RNA probe of sequence SEQ. ID. # 4;
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the RNA probe of sequence SEQ. ID. # 8;
• a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the RNA probe of sequence SEQ. ID. # 12;
• RNA probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the RNA probe of sequence SEQ. ID. # 16.
• the invention also relates to the use of at least one polynucleotide probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the FANCC gene ( accession number NM 000136.1) and / or DAD1 (accession number NM 001344.1) and / or GRIM19 (accession number NM 015965.3) and / or HADHII (accession number NM 004493.1) for manufacturing of a pharmaceutical composition intended for the treatment of psoriasis.
• the polynucleotide probe may more particularly be:
• a polynucleotide probe relating to the FANCC gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the sequence RNA probe
• a polynucleotide probe relating to the DAD1 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 6 or with the double strand cDNA probe of sequence SEQ. ID. n ° 7 or with the RNA probe of sequence SEQ. ID. # 8; a polynucleotide probe relating to the GRIM19 gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 10 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 11 or with the sequence RNA probe
• a relative polynucleotide probe having the HADHII gene which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 14 or with the double strand cDNA probe of sequence SEQ. ID. n ° 15 or with the RNA probe of sequence SEQ. ID. # 16.
• a clinical study carried out in the presence of healthy volunteers and patients suffering from chronic vulgar plaque psoriasis has made it possible to demonstrate the involvement of the genes FANCC, DAD1, GRIM19 and HADHII in psoriasis.
• Skin biopsies are taken from healthy volunteers, as well as from psoriasis plaques and non-lesional areas that appear clinically healthy, from patients with psoriasis.
• the presence or absence of psoriasis on the area of biopsied skin, as well as the severity of the psoriasis plaque, are determined by the evaluation of local clinical parameters (erythema, scaling and plaque elevation scores). psoriasis).
• the analysis of gene expression is then carried out from each individual biopsy.
• the comparison of the gene profiles corresponding to healthy skin and psoriatic non-lesional and lesional skin, makes it possible to identify genes whose expression is induced in the disease.
• the determination of gene profiles is based on the hybridization of a probe single-stranded cDNA complex (generated by reverse transcription of mRNAs from each skin biopsy) onto cDNA fragments of interest, which are deposited on a support such as a nylon filter.
• the hybridization signals obtained are proportional to the quantity of each labeled cDNA in the probe and reflect the level of expression of each mRNA in the analyzed skin sample (Nguyen C et al. Genomics (1995 Sep) 1; 29 (1): 207-16).
• Filters developed by the Applicant, specific to dermatology have been developed. These filters are made up of 474 cDNAs involved in the physiological processes of human skin. In parallel, commercial filters with 4200 human cDNAs (Invitrogen GF211) were used. These cDNA filters will make it possible to determine the expression of genes of interest in healthy skin as well as in non-lesional and lesional skin of patients suffering from chronic vulgar plaque psoriasis.
• the measurement of gene expression in healthy skin and in psoriatic skin is carried out using the technology of cDNA filters (“arrays”) on nylon support.
• the radiolabelled cDNA probes are generated by reverse transcription of the total RNAs (Invitrogen protocol), isolated from the different biopsies taken from 10 healthy volunteers and 13 subjects suffering from chronic plaques of vulgar psoriasis (RNEasy protocol, Qiagen).
• the hybridization signals obtained are proportional to the quantity of each labeled cDNA present in the probe, which hybridizes on the cDNA which is complementary to it on the filter and thus reflect the transcriptional activity of the mRNAs in the biopsies analyzed.
• the hybridization signals are measured by phospholuminescence, then quantified using the image analysis software Imagen (Biodiscovery).
• Imagen image analysis software
• the background noise linked to non-specific hybridization is measured on each filter on virgin areas of cDNA deposition (GF211 filters), or within a surrounding crown. each spot (dedicated filters).
• a detection threshold making it possible to discriminate a measurement pertaining to a real signal or noise, is automatically determined for each filter using the method described previously by Fogel et al., 2002.
• the hybridization signals are normalized by the median of the set of signals obtained for each biopsy (filters GF211), or by the expression of an RNA. exogenous messenger, serving as a reference, the expression of which is invariant between the samples analyzed.
• This exogenous transcript is added in constant quantity to the total RNA preparations from the different samples, before synthesis of the radiolabelled cDNA probe.
• the target sequence of this exogenous transcript is deposited on each filter.
• an average reference expression is determined on all the biopsies of healthy subjects, then subtracted from the expression obtained in each biopsy of non-lesional and lesional psoriatic skin (values in log). The ratio between the expression of each gene in psoriatic skin compared to healthy skin is then obtained by taking the base 10 exponential of the value calculated previously (Tables 1 to 4).
• Tables 2 and 3 indicate that these overexpressions are observed whatever the predominant type of plaques expressed in the patient (symmetrical or truncated) and also independently of the location of the psoriasis plaques.
• the abbreviations mentioned in these tables represent the location of the biopsies taken from the body for each psoriatic subject (SP) and have the following signifi cation: JG: left leg, JD: right leg, BD: right arm, BG: left arm , T: trunk.
• the different local clinical scores used to define the presence or absence of psoriasis on the biopsied skin area, as well as the severity of the psoriasis plaque, are the erythema, scaling and plaque elevation scores. These scores are conventionally measured using the following rankings:
• the measurement of erythema is done using the following classification:
• the plate elevation is measured using the following classification:
• the results of table 4 made it possible to demonstrate at the molecular level, an overexpression of the genes FANCC, DAD1, GRIM19 and HADHII in certain non-lesionne psoriatic skin.
• the measurement of the expression of the FANCC, DAD1, GRIM19 and HADHII genes therefore makes it possible to carry out a very early diagnosis of psoriasis, since the expression of these genes makes it possible to differentiate at the molecular level non-lesionneile skins already affected, while the clinical signs do not allow it.

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