WO2004007764A2 - Genes des chromosomes 3,5 et 11 impliques dans la canitie precoce - Google Patents
Genes des chromosomes 3,5 et 11 impliques dans la canitie precoce Download PDFInfo
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- WO2004007764A2 WO2004007764A2 PCT/FR2003/002153 FR0302153W WO2004007764A2 WO 2004007764 A2 WO2004007764 A2 WO 2004007764A2 FR 0302153 W FR0302153 W FR 0302153W WO 2004007764 A2 WO2004007764 A2 WO 2004007764A2
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- genes
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- gene
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- human chromosome
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q5/00—Preparations for care of the hair
- A61Q5/06—Preparations for styling the hair, e.g. by temporary shaping or colouring
- A61Q5/065—Preparations for temporary colouring the hair, e.g. direct dyes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/606—Nucleosides; Nucleotides; Nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/04—Antipruritics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q5/00—Preparations for care of the hair
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/148—Screening for cosmetic compounds
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the second obstacle in the implementation of reverse genetics methods concerns the exact definition of the phenotype.
- a perfect definition of the phenotype studied is indeed necessary.
- the choice and the composition of the sample used in the present invention were carried out according to a rigorous protocol for the allocation of the phenotype and the selection of families.
- the phenotype "premature canities" was attributed to the only individuals who presented white hair before 25 years and whose half of the hair was gray at 30 years.
- premature canities have a multigenic and not a monogenic origin, and that, on the other hand, environmental factors have an influence on the phenotype.
- the object of the present invention relates on the one hand to the genes of the chromosomal regions identified and on the other hand to the use of derived products, such as transcription or translation products, in the fields of cosmetics, therapeutics and diagnosis.
- the present invention successively relates to the use of polynucleotides deriving from a gene included in a chromosomal region of the invention, the use of agents capable of modifying the function attached to this gene, l use of the expression products of this gene and the use of agents capable of modifying the function of these expression products.
- the joint or combined use of at least two of the preceding products may prove to be judicious, in particular in the therapeutic field.
- the present invention also relates to a method for the diagnosis of premature canities based on allelic variations within the genes included in the chromosomal regions of the invention. In terms of diagnosis, it may also be particularly relevant to combine the information coming from different genes within the chromosomal areas of the invention.
- polynucleotide fragment any molecule resulting from the linear chain of at least two nucleotides, this molecule being able to be single-stranded, double-stranded or three-stranded. It can therefore be a double-stranded DNA molecule, a single-stranded DNA, an RNA, a single-stranded DNA-RNA duplex, a DNA-RNA triplex or any other combination.
- the polynucleotide fragment can be natural isolated, recombinant or else synthetic. When the polynucleotide fragment has complementary strands, the complementarity is not necessarily perfect, but the affinity between the different strands is sufficient to allow the establishment of a stable Watson Crick-type bond between the two strands.
- base pairing is preferably of the Watson-Crick type, other types are not excluded, such as a Hoogsteen or reverse Hdogsteen type pairing.
- sequence S of a molecule "corresponds" to the sequence of a given DNA molecule if we can deduce the chain of the bases of S from that of the DNA molecule given by one of the following processes 1. by identity, or 2. by identity but by changing all or part of the Thymines to Uracil, or
- two sequences remain “corresponding” if less than one error in 10 is introduced overall in one of the preceding processes (complementarity or identity, with or without T, U exchange), preferably less of an error out of 100. Consequently, the two molecules also necessarily have similar lengths, the maximum variation in length being 10% according to the accepted error rate, they preferably have a difference in length less than 1 %.
- RNA sequence resulting from the translation of any DNA molecule, "corresponds" to the sequence of this DNA molecule.
- a synthetic sequence for example hybrid DNA-RNA, may correspond to a DNA sequence. The same is true between a DNA sequence and the antisense RNA targeting this sequence.
- sequence S of a DNA molecule "corresponds" to the sequence of a given DNA molecule if we can deduce the sequence S from that of the given DNA molecule by process 1 or 3 only.
- process 1 or 3 the same latitude is allowed regarding the possibility of introducing errors in these processes, that is to say that it is considered that two DNA sequences remain
- the expression products of a DNA fragment encompass all the molecules translating the genetic information carried by this fragment.
- the RNAs corresponding to the transcription of the DNA fragment, at all stages of maturation, are therefore products of expression; the same is true for the polypeptides, at all stages of maturation, resulting from the translation of the RNAs. If cleavages occur within the polypeptide, such as, for example, cleavage of address signals, all of the resulting polypeptides are also considered to be products of expression of the original DNA fragment.
- the primary "function" of a DNA fragment is preferably to be transcribed and then translated into protein. The secondary function of DNA can be assimilated to the function of the protein resulting from the translation of this DNA.
- a DNA fragment also has other meanings in the present invention.
- a DNA fragment can belong to a regulatory region of a gene, its function is then either to be the site of binding of enhancers or inhibitors, or else to be the site of binding RNA polymerase, or to be a recognition site for the positioning of RNA polymerase, or any other function generally assimilated to a regulatory sequence.
- a genetic marker is a detectable DNA sequence.
- markers are specific sequences of DNA that can take different forms depending on the individual. This polymorphism of markers makes it possible to follow their transmission within the framework of family trees.
- SNP Single nucleotide polymorphism
- microsatellite markers are tools for locating the genes involved in premature canities. As there is much less polymorphism in genes than in markers, a gene allele will be represented by several alleles of the same microsatellite marker.
- the physical unit of measure is the number of base pairs.
- the centimorgan is often used, it is a unit of recombination therefore a unit of genetic measurement and not physical.
- Two particular sequences of the same chromosome are one centimeter apart if they recombine once in a hundred during meiosis.
- One centimorgan is approximately 10 6 base pairs.
- Another method for locating particular DNA sequences along the chromosomes is to define their position relative to markers dotting the chromosomes and whose position is perfectly determined and known. Markers widely used are microsatellite markers of which there are very complete maps.
- the GDB "Genome Database” is a database known worldwide for listing, among other things, the STSs (Sequence tagged sites), specific and unique bounds of DNA of which microsatellites are a part.
- the DxSxxxx codes (for example D6S257), used to identify these markers, are their accession number in GDB. These codes are an unambiguous and universal means of identification because only GDB assigns this type of code.
- a SNP Single Nucleotide Pholymorphism
- SNP Single Nucleotide Pholymorphism
- chromosomal region between two markers is meant the entire sequence between these two markers, the limits being understood, therefore the sequence of markers including.
- the indices making it possible to locate a gene come from the comparison of the transmission of a phenotype, supposedly induced by a mutated gene or by a given allele, with the transmission of known markers, within the same family. These phenotype and marker co-segregation data make it possible to establish a genetic linkage analysis.
- Binding is determined by analyzing the transmission pattern of a gene and a marker in families that lend themselves to it. Binding analysis is based on the co-transmission of certain forms of markers with the defective or modified form of a gene. But it is an indirect analysis in the sense that on the one hand, during a first stage, a phenotype is associated with the defective or modified form of the gene. An error in the assignment of some phenotypes distorts the study.
- the result of the linkage analyzes obviously depends on the degree of linkage between the marker and the locus of the disease.
- Five centimorgans (5 cM) is considered a minimum of binding for a diagnosis.
- a binding to 5 cM means that there is a 95% chance of arriving at a correct conclusion and only 1 in 20 chances that a recombination has occurred between the marker and the locus of the disease.
- gene in the. framework of the present invention, is meant not only the strictly coding part, but also the non-coding parts such as the introns, and the regulatory parts, in 5 ′ and 3 ′, the UTRs (UnTranslated Region), in particular the promoter (s) , "Enhancers” etc ... associated. , ;
- the inventors have identified 3 distinct chromosomal regions, belonging to chromosomes 3, 5 and 11 and which are involved in premature canities. These 3 regions are each of the chromosomal regions or zones of the invention. The inventors have more particularly highlighted the involvement of certain genes belonging to these chromosomal regions, called genes of the invention.
- the invention relates to the genes KIAA1042, CCK-, CACNA1D, ARHGEF3 and AL133097 of the human chromosome 3, identified by the inventors as being involved in premature canities and the uses of products derived from these genes, such as transcription or expression products.
- These genes belong to the first chromosomal zone of the invention which is delimited on chromosome 3 by the microsatellite markers D3S1277 and D3S1285. This area will more particularly be called “first chromosomal area of the invention”.
- the 5 genes mentioned will be more particularly called the 5 genes of the invention of chromosome 3.
- the invention relates to the genes KLHL3, HNRPAO,
- CDC25C EGR1, C5orf6, C5orf7, LOC51308, ETF1, HSPA9B, PCDHAI to PCDHA13, CSF1R, RPL7, PDGFRB, TCOF1, AL133039, CD74, RPS14, NDST1, G3BP, GLRA1, C5orf3, MFTI1 chromopn, and by the inventors as being involved in early canities and the uses of products derived from these genes, such as transcription or expression products.
- These genes belong to the second chromosomal zone of the invention which is delimited on chromosome 5 by the microsatelhtes markers D5S2115 and D5S422. This area will be more particularly called “second chromosomal area of the invention”.
- the genes mentioned will be more particularly called the genes of the invention of chromosome 5.
- the invention relates to the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300 chromosome identified by the inventors as being involved in premature canities and the uses of products derived from these genes, such as transcription or expression products.
- These genes belong to the third chromosomal zone of the invention which is dehmited on chromosome 11 by the microsatellite markers D11S898 and D11S925. This zone will more particularly be called "third chromosomal zone of the invention".
- the 18 genes mentioned will be more particularly called the 28 genes of the invention of chromosome 11.
- the present invention covers polynucleotide fragments having a minimum length of 18 nucleotides, corresponding at least partially to one of the genes of the invention, these DNA fragments having the functional characteristic of 'be involved in canities, or in premature canities, and possibly in both phenomena.
- a fragment involved in canities or early canities and having a sequence meeting the requirements mentioned above can be used in therapy.
- the first aspect of the invention relates to the genes of human chromosome 3 identified by the inventors as being involved in premature canities.
- a fragment as covered by the invention has a sequence corresponding to all or part of a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097. These genes are included in the first chromosomal area of the invention which is delimited on chromosome 3 by the microsatelhtes markers D3S1277 and D3S1285.
- the second aspect of the invention relates to the genes of human chromosome 5 identified by the inventors as being involved in premature canities.
- a fragment as covered by the invention has a sequence corresponding to all or part of a gene from human chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETF1, HSPA9B , PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRA1, C5orf3, MFAP3, GALNTIO and FLJ11715.
- These genes are included in the second chromosomal zone of the invention which is delimited on chromosome 5 by the microsatellite markers D5S2115 and D5S422.
- the third aspect of the invention relates to the genes of human chromosome 11 identified by the inventors as being involved in premature canities.
- a fragment as covered by the invention has a sequence corresponding to all or part of a gene from human chromosome 11 chosen from the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536 , FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650.
- the polynucleotide fragment referred to in the context of the invention corresponds to a fragment of a chromosome. This fragment has a minimum length of 18 nucleotides, and a maximum length which can go up to the total length of the gene in question, or of several genes of the invention. Preferably, the fragment has a number of nucleotides greater than 18. A particularly preferred length is between 18 and 10,000 nucleotides, preferably between 30 and 8000 nucleotides.
- fragments whose length is between 30 and 5000 nucleotides, preferably between 50 and 3000 nucleotides, for example between 100 and 2000 nucleotides, or between 200 and 1000 nucleotides.
- the invention also relates to the use in cosmetics or in therapy of a polynucleotide fragment or of the expression product of a fragment or of an agent modulating the function of a fragment, or else of an agent modulating the function of the expression product of a fragment, where the fragment in question corresponds to all or part of a gene from chromosome 11 chosen from the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535 , DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650, or all or part of a gene from chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETFB, HSP PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOF
- products of the invention the fragment, the expression product of a fragment, the agent modulating the function of a fragment, and the agent modulating the function of expression of a polynucleotide fragment corresponding to all or part of one of the genes of the invention.
- the present invention relates first of all to uses in the field of cosmetics.
- Cosmetic means any application which only tends to modify the aesthetic and has no therapeutic aim.
- the product of the invention can be packaged in different suitable forms, alone or in combination with other agents.
- preferred forms are intended for local applications and they relate to creams, lotions, gels, emulsions, ointments and shampoos.
- Other forms can also be envisaged for uses according to the invention, in particular in the form of pills for oral administration.
- a particularly preferred field is that of pigmentation.
- the pigmentation can be that of the skin or of the integuments. It can be the color of the pigmentation as well as the absence of pigmentation; problems relating to the quality and intensity of pigmentation are also concerned with the present invention.
- the subject of the invention relates to the use of at least one product of the invention to prevent and / or limit and / or stop the development of canities.
- the object of the invention also relates to the use of at least one product of the invention to promote the natural pigmentation of hair and / or gray hair.
- Another object of the present invention relates to a cosmetic treatment process for canities characterized in that a composition comprising at least one product of the invention is applied to the area to be treated.
- the invention also relates to a cosmetic treatment process intended to promote the natural pigmentation of gray and white hair and / or body hair, characterized in that a composition comprising at least one product of the invention is applied to the area to be treated . . -; - •
- the areas to be treated can be, for example and without any limitation, the scalp, the eyebrows, the mustache and / or the beard. . ⁇ -. • More particularly, the methods for treating canities and natural pigmentation of gray or white hair and / or hair consist in applying a composition comprising at least one product of the invention.
- the treatment methods for combating canities and / or for stimulating the natural pigmentation of gray and white hair and / or body hair may for example consist in applying the composition to the hair and the scalp, in the evening, keeping the entire composition. at night in contact and optionally carry out a shampoo in the morning or wash the hair using this composition and leave it in contact again for a few minutes before rinsing.
- the composition according to the invention has proved to be particularly advantageous when it is applied in the form of a hair lotion, optionally rinsed or even in the form of a shampoo.
- the present invention relates to therapeutic uses in the field of pigmentation.
- the type of pigmentation which must be modified relates to the pigmentation of the integuments, in particular the nails or the hairs.
- the pigmentation whose characteristics one seeks to modify is that of the hair system in general and of the hair, mustache and eyebrows in particular.
- the present invention makes it possible to modify the phenomenon of stopping the pigmentation of the hair, that is to say, canities, in particular when the latter occurs prematurely in a person and when we speak of premature canities.
- the active products used in the composition of a medicament are preferably associated with pharmaceutically acceptable excipients.
- All the routes of administration considered acceptable can be used within the framework of the invention, in particular intradermal, intravenous, muscular, oral, otic, nasal, optical route.
- the formulation is preferably adapted to the chosen route of administration.
- the uses for the manufacture of a medicament according to the invention can include other active ingredients in their formulation.
- administration of a medicament as defined in the invention can be combined with the administration of another medicament, whether this administration is simultaneous, sequential or separate.
- the various products which are used within the framework of uses in therapy can be combined and enter into the composition of a single medicament or else can be used for the manufacture of different medicaments.
- they form part of the composition of separate drugs they can be administered at different frequencies.
- the preferred characteristics and variants of the products which are used in the uses according to the invention may be identical in the context of uses in cosmetology and for uses of the same product in the manufacture of a medicament.
- the use of products according to the invention may require that the product be introduced into a body fluid, or into tissues, or into cells.
- the product is active in the cytoplasm of the cells, or else in the cell nucleus.
- the first use, cosmetic or therapeutic, envisaged in the context of the invention is the use of a polynucleotide fragment whose sequence corresponds, at least in part, to one of the genes of the invention.
- the polynucleotide fragment is used in the manufacture of a medicament.
- this polynucleotide fragment can be a DNA molecule, single or double strand, circular or linear, an RNA molecule drunk from any other molecule envisaged in the definition of polynucleotide fragment given above.
- this fragment 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 fragment in its genome. It can also be an isolated molecule.
- the fragment is preferably under the control of regulatory sequences.
- said vector preferably comprises all the sequences necessary for transcription and optionally translation of the fragment.
- This fragment can also be surrounded by flanking regions allowing a step of homologous recombination with another polynucleotide fragment, possibly leading to the insertion of the fragment of the invention in the genomic DNA of a target cell.
- the polynucleotide fragment as described may be in natural form or else be of a synthetic nature, or else be partly one and partly the other, in particular if it is a “duplex” molecule constituted of two strands with different origins.
- the polynucleotide fragment can be isolated, it can have undergone a purification step. It can also be a recombinant fragment, for example synthesized in another organism. According to a preferred example, it is a DNA fragment which has been amplified by PCR (Polymerization Chain Reaction) and then purified.
- the first use makes use of a polynucleotide fragment associated with a probe.
- This characteristic can make it possible, among other things, to follow the location of the fragment, 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.
- the probe can also make it possible to follow the degradation of the fragment.
- the nature of the probe is preferably fluorescent, radioactive or enzymatic. Those skilled in the art will know which probe is best suited as a function of the characteristic that they want to be able to follow.
- polynucleotide fragment which is used in the context of this first use according to the invention, can be used in a hybridization test, sequencing, microsequencing or else a test for detection of mismatching.
- This fragment according to the invention contains at least 18 successive nucleotides, these
- a fragment according to the invention may contain only 18 bases complementary to 18 successive bases of one of the genes of the invention.
- the fragment described can be the cDNA or the RNA of one of the genes previously described. It can correspond to one or more exons of one of the genes of the invention, it can correspond to a regulatory sequence of one of these genes identified on chromosomes 3, 5 and 11.
- the number of polynucleotide fragments as defined above is not limited and is not necessarily limited to one.
- polynucleotide fragments whose sequence corresponds, at least in part, to one of the genes of the invention within the chromosomal regions of the invention.
- sequences of the different fragments correspond to distinct genes of the invention or to distinct exons.
- This first use according to the invention is preferably in the field of cosmetics.
- the first use described involves a genetic modification, whether it is induced by a nucleotide fragment as described or not.
- this first use according to the invention makes it possible to restore the function of this gene by introducing a polynucleotide fragment which represents a new wild copy of the defective endogenous gene.
- this first use according to the invention makes it possible to abolish the function of this gene by introducing an antisense RNA which will block the translation of said gene.
- a second use envisaged by the present invention is the use of an agent modulating the function of a DNA fragment corresponding at least in part to a gene chosen from the genes of the invention.
- the agent thus defined is used in the manufacture of a medicament.
- the DNA fragment has at least 18 nucleotides.
- Such an agent according to the invention may be capable of modulating the function of an exogenous DNA fragment of which part of the sequence corresponds to one of the genes of the invention identified by the inventors, or else it may be capable modulate the function of an endogenous sequence included in one of these genes of the invention.
- an agent serving for this second use according to the invention modulates not only the function of an exogenous DNA fragment as defined, but also of the corresponding endogenous DNA fragment.
- the DNA fragment whose function is modulated can partially correspond to one of the 5 genes of the invention of chromosome 3.
- the DNA fragment whose function is modulated can partially correspond to one of the genes of the invention of chromosome 5, or one of the 18 genes of the invention of chromosome 11.
- it may be a plasmid having only a short sequence corresponding to one of the genes mentioned.
- the sequence correspondence is established on at least 18 successive nucleotides.
- modulating such a function consists in promoting or inhibiting the capacity of said fragment to be transcribed. It can also consist in modifying the site of initiation and termination of transcription, or else in modifying the rate of initiation of transcription. In another case, modulating the function can also consist in modifying the splicing of the RNA, for example by modifying the DNA recognition signals responsible for the distribution between introns and exons.
- modifying its function can consist of inhibiting the binding of enhancers or inhibitors. On the contrary, it may consist in promoting their fixation, or else in promoting the fixation of other transcription factors. It is the same when it comes to sequences used by RNA polymerase.
- the number of products, in this case agents modulating the function of a DNA fragment corresponding at least in part to one of the genes of the invention is not limited and may be greater than one.
- the various agents which are used have in common all modulating the function of DNA fragments whose sequence belongs to or corresponds to at least part of the same chromosomal region of l 'invention.
- this region is that of the human chromosome 3 identified by the inventors.
- the chromosomal region in question is that identified by the inventors on chromosomes 5 and 11 respectively.
- the different agents can modulate the same gene from one of the regions, or else different genes within a region of the invention.
- Agents according to the invention are, for example, single-stranded DNA molecules capable of binding to defined subregions of one of the genes of the invention, to form triple helices. Under these conditions, agents according to the invention abolish the function of the sub-region to which they hybridize.
- agents according to the invention are preferably polypeptides capable of interacting with defined sub-regions of one of the genes of the invention.
- agents according to the invention are enhancers or inhibitors which bind to regions regulators of one of the genes of the invention of human chromosome 3, or of one of those of human chromosome 5, or of one of those of chromosome 11.
- Another category of agents according to the invention relates to molecules capable of interacting with precise regions of DNA to change its conformation.
- Another category relates to molecules interacting with inhibitors or enhancers to modify their function, inhibitors or enhancers having the initial function of modifying the expression of DNA fragments belonging to one of the genes of the invention.
- An agent which is used according to this second use of the invention can in particular modulate the function of a DNA fragment corresponding to 18 successive bases of one of the genes previously described.
- This second use according to the invention is preferably in the field of cosmetics. This use can also allow the manufacture of a medicament for a therapeutic action, in the field of pigmentation. ",
- the second use described involves a genetic modification, whether it is induced by an agent modulating the function of a DNA fragment as described or not.
- this second use according to the invention makes it possible to abolish the function of this gene by introducing an agent which will block the translation of said gene by binding, for example , to its promoter region.
- this second use according to the invention makes it possible to restore the function of this gene by introducing an agent which will activate the transcription of said gene, for example by binding to its promoter region, or else by attaching itself to a possible inhibitor which will thereby cease inactivating said gene.
- a third use envisaged by the present invention is the use of an agent modulating the function of the expression product of a DNA fragment corresponding at least in part to one of the genes of the invention.
- the agent thus defined is used in the manufacture of a medicament.
- the DNA fragment has at least 18 nucleotides.
- an agent according to the invention modulates the function of a transcript derived from a DNA fragment corresponding at least in part to one of the 5 genes of the invention of chromosome 3.
- an agent according to the invention modulates the function of a polypeptide resulting from the translation of one of the transcripts mentioned.
- the DNA fragment whose function of the expression product is modulated can correspond at least in part to a gene of the invention of chromosome 5, or else to a gene of the invention of chromosome 11.
- Such an agent according to the invention may be capable of modulating the function of the expression product of an exogenous DNA fragment of which part of the sequence corresponds to one of the genes of the invention identified by the inventors, or although it may be able to modulate the function of the expression product of an endogenous sequence of one of these genes of the invention.
- an agent serving for this third use according to the invention modulates not only the function of an expression product of an exogenous DNA fragment as defined, but also of the corresponding endogenous DNA fragment.
- Modulating the function of a polypeptide can be achieved in different ways. In particular, it is possible to increase or decrease its activity, its yield, its specificity, its greed for an antibody, it is possible to modify its substrate for an enzyme, its conversion rate.
- Agents according to the invention are preferably RNA molecules, called antisense RNA, which hybridize with at least one transcript derived from a DNA fragment corresponding at least in part to one of the genes of the invention.
- Other agents fulfilling the same roles can be single-stranded DNA molecules, or hybrid DNA-RNA molecules.
- the role of these agents according to the invention is preferably to promote, prevent, delay, accelerate or introduce errors in the translation of said transcript.
- agents according to the invention belong to the class of polypeptides.
- the invention relates to proteins capable of binding to said transcript and thus of modulating its translation.
- agents from the class of polypeptides can be of natural or synthetic origin (synthesized chemically or by biotechnology). In particular, they may be antibodies.
- this modulation can result in the action of promoting, preventing, delaying, accelerating or introducing errors in the translation of said transcript.
- the interaction between the polypeptide and said transcript can constitute an obstacle to the normal fixation of ribosomes.
- Agents as defined in the present invention can modulate the function of the protein encoded by a DNA fragment corresponding at least in part to one of the 5 genes of the invention of chromosome 3 or one of the genes of l invention of chromosome 5 or one of the 18 genes of invention of chromosome 11. These agents may or may not be protein in nature.
- An agent according to the invention can intervene at an early stage by preventing the correct folding of the protein.
- An agent according to the invention can also modify the function of said protein by modifying its three-dimensional structure after folding. It is also conceivable that this agent is an inhibitor of the protein, in particular a competitive inhibitor.
- the agents which may be suitable in the context of the present invention are not limited to those mentioned above. *
- the number of agents modifying the function of an expression product as defined above is not limited and is not necessarily limited to one.
- the various agents which are used have in common all modulating the function of expression products of DNA fragments whose sequence belongs to or corresponds to at least part of a gene from the same chromosomal region of the invention.
- this region is that of the human chromosome 3 identified by the inventors.
- the chromosomal region in question is that identified by the inventors on chromosomes 5 and 11 respectively.
- This third use according to the invention is preferably in the field of cosmetics. This use can also allow the manufacture of a medicament for a therapeutic action, in the field of pigmentation.
- the third use described involves a genetic modification, whether it is induced by an agent modulating the function of the expression product of a DNA fragment as described or not.
- this third use according to the invention makes it possible to abolish the function of this gene by introducing an antisense RNA which will block the translation of said gene by preventing the RNA-protein passage.
- Another preferred situation consists in choosing for agent an antibody capable of binding to the protein resulting from the translation of said gene.
- a fourth use envisaged by the present invention, in the field of therapy and in that of cosmetics, is the use of an expression product of a DNA fragment corresponding at least in part to one of the genes of the invention.
- the agent thus defined is used in the manufacture of a medicament.
- the DNA fragment has at least 18 nucleotides.
- such an expression product is the RNA transcript derived from a DNA fragment corresponding at least in part to one of the 5 genes of the invention of chromosome 3, or else one of the genes of l he invention of chromosome 5, or one of the 18 genes for the invention of chromosome 11, whatever the stage of maturation of said transcript.
- the transcript may therefore be smaller than the DNA fragment from which it originates.
- the expression product is an RNA molecule, it contains at least 18 nucleotides.
- an expression product according to the invention results from the translation of one of the transcripts mentioned.
- Such an expression product can therefore contain less than 6 amino acids if the transcript from which it is derived has undergone splicing steps.
- a peptide expression product contains at least 6 amino acids.
- an expression product used according to the invention does not necessarily come from the steps of transcription or translation of genomic DNA.
- an expression product used according to the invention may be an expression product derived from exogenous DNA of which at least part of the sequence corresponds to a part of one of the genes of the invention.
- Expression products as used by the present invention are preferably RNA molecules, called antisense RNA, which hybridize with at least one transcript derived from a DNA fragment corresponding at least in part to the one of the genes of the invention.
- antisense RNAs targeting a specific RNA it is possible to use RNAs derived from the transcription of the same DNA sequence as the target but not from the leader strand, but from its sequence complementary carried by the other strand. RNA fragments are thus obtained complementary to the target fragments normally synthesized by the cell.
- the expected role of these expression products according to the invention is preferably to promote, prevent, delay, accelerate or introduce errors in the translation of the transcripts normally synthesized by the cell.
- Other preferred expression products according to the invention belong to the class of polypeptides.
- the invention relates to proteins capable of introducing a change in the functioning of the cell in which they act.
- the number of products of expression of a DNA fragment whose sequence belongs to or corresponds at least in part to one of the genes of the invention is not limited and may be greater than one.
- the various products which are used have in common that they are all products of expression of DNA fragments whose sequence belongs to or corresponds to at least part of a gene. of the same chromosomal region of the invention.
- this region is that of the human chromosome 3 identified by the inventors.
- the chromosomal region in question is that identified by the inventors on chromosomes 5 and 11 respectively.
- This fourth use according to the invention is preferably in the field of cosmetics. This use can also allow the manufacture of a medicament for a therapeutic action, in the field of pigmentation.
- the fourth use described involves a genetic modification, whether it is induced by an expression product of a DNA fragment as described or not.
- this fourth use according to the invention makes it possible to abolish the function of this gene by introducing an antisense RNA which will block the translation of said gene by fixing itself , to the transcript synthesized by the cell.
- this fourth use according to the invention makes it possible to restore the function of this gene by introducing into the cell or else an RNA molecule allowing the synthesis of the protein. encoded by the gene or the protein encoded by the gene.
- the cosmetic uses are preferably in the field of pigmentation.
- the product of the invention can be incorporated into a cosmetic or pharmaceutical composition.
- Such a composition comprises, in a pharmaceutically or cosmetically acceptable medium, an amount of products of the invention of between 0.001 and 10% by weight by volume.
- composition can be administered orally or applied to the skin (on any cutaneous zone of the body) and / or the scalp or the hair.
- the composition may contain the product or products of the invention in solution in a food liquid such as an aqueous or hydroalcoholic solution, optionally flavored. They can also be incorporated in a solid ingestible excipient and be presented for example in the form of granules, pills, tablets or dragees. They can also be placed in solution in a liquid food itself possibly packaged in ingestible capsules.
- a food liquid such as an aqueous or hydroalcoholic solution
- a solid ingestible excipient and be presented for example in the form of granules, pills, tablets or dragees. They can also be placed in solution in a liquid food itself possibly packaged in ingestible capsules.
- the composition may be in all the dosage forms normally used, particularly in cosmetology.
- a preferred composition of the invention is a cosmetic composition suitable for topical application to the scalp and / or the skin.
- the composition which can be used can in particular be in the form of an aqueous, hydroalcoholic or oily solution or of dispersion of the lotion or serum type, of emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of '' an oily phase in an aqueous phase (O / W) or vice versa (W / O), or suspensions or emulsions of soft consistency of the aqueous or anhydrous cream or gel type, or alternatively of microcapsules or microparticles, or of vesicular dispersions of ionic and / or non-ionic type.
- ointment can thus be in the form of an ointment, a tincture, a cream, an ointment, a powder, a patch, a soaked tampon, a solution, an emulsion or a vesicular dispersion, a lotion, a gel, a spray, suspension, shampoo, aerosol or foam.
- ointment can be in the form of an ointment, a tincture, a cream, an ointment, a powder, a patch, a soaked tampon, a solution, an emulsion or a vesicular dispersion, a lotion, a gel, a spray, suspension, shampoo, aerosol or foam.
- They can be anhydrous or aqueous. It can also consist of solid preparations constituting soaps or cleaning bars.
- compositions are prepared according to the usual methods.
- the composition may in particular be a composition for hair care, and in particular a shampoo, a styling lotion, a treatment lotion, a styling cream or gel, a composition of dyes (in particular oxidation dyes) optionally in the form of coloring shampoos, restructuring hair lotions, masks.
- the composition will preferably be a cream, a hair lotion, a shampoo or a conditioner.
- the amounts of the various constituents of the compositions which can be used are those conventionally used in the fields under consideration.
- the proportion of the fatty phase can range from 5% to 80% by weight, and preferably from 5% to 50% by weight relative to the total weight of the composition.
- the oils, waxes, emulsifiers and co-emulsifiers used in the composition in the form of an emulsion are chosen from those conventionally used in the cosmetic field.
- the emulsifier and the co-emulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5 to 20% by weight relative to the total weight of the composition .
- the emulsion can, in addition, contain lipid vesicles.
- the fatty phase can represent more than
- the composition will be such that the product or products of the invention are encapsulated in a coating such as microspheres, nanospheres, oleosomes or nanocapsules, the coating will be chosen according to the chemical nature of the product of the invention.
- the microspheres can be prepared according to the method described in patent application EP 0 375 520.
- the nanospheres can be in the form of an aqueous suspension and can be prepared according to the methods described in patent applications FR 0015686 and FR 0101438.
- Oleosomes consist of an oil-in-water emulsion formed by oily globules provided with a lamellar liquid crystal coating. dispersed in an aqueous phase (see patent applications EP 0641 557 and EP 0705 593).
- the products of the invention can also be encapsulated in nanocapsules consisting of a lamellar coating obtained from a silicone surfactant (see patent application EP 0 780 115), the nanocapsules can also be prepared based on polyesters hydrodispersible sulfonic (see patent application FR 0113337).
- the products of the invention may also be complexed on the surface of cationic oily globules, whatever their size (see patent applications EP 1 010 413, EP 1 010 414, EP 1 010 415, EP 1 010 416, EP 1 013 338 , EP 1 016453, EP 1 018 363, EP 1 020 219, EP 1 025 898, EP 1 120 101, EP 1 120 102, EP 1 129 684, EP 1 160005 and EP 1 172 077).
- the products of the invention can finally be complexed on the surface of nanocapsules or nanoparticles provided with a lamellar coating (see EP 0447 318 and EP 0 557489) and containing a cationic surfactant on the surface (see the references cited above for cationic surfactants).
- compositions such as the coating of the product or products of the invention will have a diameter less than or equal to 10 ⁇ m.
- the composition can also contain adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, filters, odor absorbers and coloring matters.
- adjuvants customary in the cosmetic field such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, filters, odor absorbers and coloring matters.
- the amounts of these various adjuvants are those conventionally used in the cosmetic field, and for example from 0.01% to 10% of the total weight of the composition.
- These adjuvants depending on their nature, can be introduced into the fatty phase, into the aqueous phase and / or into the lipid spherules.
- solvents which can be used, mention may be made of lower alcohols, in particular ethanol and isopropanol, propylene glycol.
- hydrophilic gelling agents which can be used, mention may be made of carboxyvinyl polymers (carbomers), acrylic copolymers such as acrylate / alkyl acrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays, and, as gelling agents lipophilic- mention may be made of modified clays such as bentones, metal salts of fatty acids such as aluminum stearates and hydrophobic silica, ethylcellulose, polyethylene.
- the compositions can combine at least one product of the invention with other active agents.
- active agents there may be mentioned by way of example:
- agents modulating the differentiation and / or proliferation and / or pigmentation of skin cells such as retinol and its esters, vitamin D and its derivatives, estrogens such as Toestradiol, cAMP modulators such as POMC derivatives, adenosine, or forskolin and its derivatives, prostaglandins and their derivatives, triiodotrionine and its derivatives; ;
- - extracts of microorganisms - anti-free radical agents such as -tocopherol or its esters, superoxide dismutases or its mimetics, certain metal chelators or ascorbic acid and its esters;
- anti-seborrhoeics such as certain sulfur amino acids, 13-cis retinoic acid, cyproterone acetate; - other agents for combating scalp conditions such as zinc pyrithione, selenium disulfide, climbazole, undecylenic acid, Ketoconazole, lapiroctone olamine (octopirox) or ciclopiroctone (ciclopirox); in particular, it may be active stimulating regrowth and / or promoting the slowing down of hair loss, we can more particularly cite without limitation:
- - nicotinic acid esters including in particular tocopherol nicotinate, benzyl nicotinate and C1-C6 alkyl nicotinates such as methyl or hexyl nicotinates; - pyrimidine derivatives, such as 2,4-diamino 6-piperidmopyrimidine 3-oxide or "Minoxidii” described in US patents 4,139,619 and US 4,596,812;
- antibacterial agents such as macrolides, pyranosides and tetracyclines, and in particular l ⁇ rythromycin;
- - calcium antagonist agents such as Cinnarizine, Nimodipine and Nifedipine
- - hormones such as estriol or analogues, or thyroxine and its salts
- - antiandrogenic agents such as oxendolone, spironolactone and flutamide
- - ATP-dependent potassium channel agonists such as cromakalim and nicorandil.
- the present invention relates to methods for diagnosing a predisposition to premature canities in an individual.
- premature canities is a phenotype which has been defined by the inventors as being, inter alia, characterized by the appearance of the first white hairs early in life, and preferably around the age of 18 years. As this phenotype is passed on to the next generation, it may be important for individuals with a relative or loved one to determine before symptoms appear whether or not they will be affected.
- the diagnostic method according to the invention is perfectly suited to individuals under the age of 18 years.
- a method for determining a predisposition to premature canities includes a first step of selecting one or more markers which will be used in the following steps.
- marker is meant a sequence DNA whose various allelic variations carry information.
- Such a marker can be a short sequence of a gene whose mutation is the source of the phenotype. It can also be a marker physically located on the chromosome in a region very close to a gene involved in premature canities.
- the marker is a SNP ('single nucleotide polymorphism').
- the marker or markers selected belong to the region of human chromosome 3 comprising the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097.
- the markers are chosen as belonging to the sequence of one of these genes.
- the marker or markers selected belong to the region of human chromosome 5 comprising the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETF1, HSPA9B, PCDHAI to PCDHAI 3, CSFIR, RPL7, PDGFRB, TCOFl, ALI 33039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and FLJ11715.
- the markers are chosen as belonging to the sequence of one of these genes.
- the marker or markers selected belong to the region of human chromosome 11 comprising the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2 , ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650.
- the markers are chosen as belonging to the sequence of one of these genes.
- the next step in implementing a method according to the invention consists, for the marker or markers chosen, in determining the alleles present in a sample of genetic material originating from the individual who is undergoing the diagnostic test. Two different alleles, carried by the two versions of the chromosome, can be identified.
- the sample containing the genetic material can be blood, a single drop being sufficient for the implementation of a method according to the invention.
- Other samples of body fluids can be used in the context of the invention. It is also possible to use a few cells from the individual. Those skilled in the art will be able to determine which sample can be used in the context of this test, while minimizing the inconvenience for the individual undergoing it.
- This diagnostic test could possibly be combined with other genetic tests. Current techniques, well known to the molecular biologist, can be used to determine the alleles of the marker or markers chosen; Hybridization tests are in particular very common in this kind of steps.
- markers are potentially preferred in the context of the implementation of the method of the invention.
- bi-allelic markers can prove to be particularly suitable if one allelic form reflects a predisposition to premature canities while the other allelic form reflects on the contrary the absence of such a predisposition.
- Other more common markers are polymorphic and can be found in at least two allelic forms and generally more than two.
- ar ⁇ arqueurs which can be selected at the first stage of the process of the invention, particularly well known markers are SNPs. When selecting the marker, it is very important to base yourself on the informative value of the marker polymorphism.
- a particularly privileged situation consists in selecting a marker whose certain allelic variants reflect a predisposition to premature canities while all the other variants reflect on the contrary the absence of such a predisposition.
- the marker does not entirely fulfill such a condition, that is to say that for example certain alleles are present preferentially but not exclusively in individuals predisposed to canities. In these situations, it may be very wise to select several markers, in order to establish a diagnostic test as reliable as possible.
- the markers selected are SNPs
- the different allelic variants correspond to the modification of a base.
- a particularly advantageous situation to look for when selecting the marker corresponds to the situation where certain alleles (modification of a base) are characteristic of a predisposition to premature canities.
- the marker or markers selected in the first step can be chosen from the SNPs of chromosome 3 mentioned in the summary table of Example 2.
- the marker or markers selected in the first step can be chosen from the SNPs of chromosome 5 mentioned in the summary table of Example 2.
- the marker or markers selected in the first step can be chosen from the SNPs of chromosome 11 mentioned in the summary table of Example 2.
- the methods of the invention are not limited to the two stages described and may contain other stages before or after the two stages mentioned.
- a method of the invention may comprise the additional step of comparing the allaque form of the selected marker or markers with the allaque form of this or these same markers in other individuals.
- This additional comparison step may be necessary in order to establish the diagnosis.
- it may be useful to make a comparison with the form of the marker (s) in individuals known to have premature canities and possibly also with the form of the marker (s) in individuals known to be free from such a predisposition. Since premature canities are a presumably multigenic disease, the causes of predisposition are numerous and can hardly be comprehensively considered.
- some members of which were prematurely affected by canities it is very likely that the cause of the susceptibility is unique.
- a comparison which is particularly rich in information is the comparison of the alleles of the marker of the individual undergoing the test with alleles of the same marker for people in his family whose phenotype is known. If several markers have been selected, it is preferable to repeat this operation for all the markers.
- the present invention also relates to methods of screening for molecules having a particular effect.
- the invention relates to a method of identifying molecules capable of modulating the function of a polynucleotide fragment.
- the polynucleotide fragment whose function one seeks to modulate comprises at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from human chromosome 3 chosen from. the KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097 genes.
- the polynucleotide fragment whose function is sought to modulate comprises at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene of human chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C , EGR1, C5orf6, C5orf7, LOC51308, ETFl, HSPA9B, PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO.
- the polynucleotide fragment whose function one seeks to modulate comprises at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from human chromosome 11 chosen from the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650.
- the method of identifying molecules capable of modulating the function of one or other of these fragments comprises a step of bringing the test molecule and the polynucleotide fragment into the presence. Another step in the process is the detection of a variation in a parameter linked to the function of said fragment, for example the detection of a possible fixation of this molecule on the polynucleotide fragment demonstrated by a ligand detection technique.
- the “second use according to the invention” is the use of an agent modulating the function of a DNA fragment corresponding at least in part to one of the genes of the invention.
- the screening process identifies such agents.
- Modulation of function may correspond to a decrease in the ability to be transcribed, or translated, or to a change in the ability to interact with other factors. Depending on the properties of said fragment used, a person skilled in the art is able to determine which parameter is the variation of which is easy to monitor.
- the identification method of the invention is not limited to the steps previously described, other previous or later steps can be applied.
- the present invention also covers the molecules identified by the method described above.
- the present invention covers inhibitors of the functions of the polynucleotide fragments of the invention.
- the present invention also relates to methods of screening molecules capable of modulating the function of the expression product of a polynucleotide fragment of the invention.
- the expression product whose function one seeks to modulate is that of a DNA fragment belonging to and / or corresponding to all or part of one of the 5 genes of the invention of human chromosome 3.
- the expression product whose function one seeks to modulate is that of a DNA fragment belonging and / or corresponding to all or part of one of the genes of the invention of human chromosome 5.
- the expression product whose function is sought to modulate is that of a DNA fragment belonging to and / or corresponding to all or part of one of the 18 genes of human chromosome 11.
- the DNA fragment comprises at least 18 nucleotides.
- the method for identifying molecules capable of modulating the function of the expression product of a DNA fragment as described comprises a step of bringing the test molecule into contact with the expression product. Another step of the process is the detection of a variation of a parameter linked to the function of said expression product, for example the detection of a possible fixation of this molecule on the expression product demonstrated by a technique. ligand detection.
- the expression product of a DNA fragment is understood to mean both the molecules, RNA resulting from the transcription of the fragment, at any stage of maturation, and the polypeptides resulting from translation, also at any stage of maturation.
- RNA molecule different stages of maturation are represented by the presence or absence of the cap, of polyadenylated tail, for example.
- stages of maturation of a polypeptide one includes inter alia the polypeptides before and after folding, before and after cleavage of the different addressing signals, with and without glycosylation, with and without disulfide bridges.
- the functions fulfilled by the expression products of the DNA fragments they are very numerous and they depend on the nature of the expression product in question. Examples have already been given earlier in the present application.
- the “third use according to the invention” is the use of an agent modulating the function of the expression product of a DNA fragment.
- the screening process identifies such agents.
- modulate the function of the expression product of a DNA fragment examples have already been given to define the third use according to the invention.
- the identification method of the invention is not limited to the steps previously described, other previous or later steps can be applied.
- the present invention also covers the molecules identified by the method described above. In particular, the present invention covers inhibitors of the functions of the expression products of the polynucleotide fragments of the invention.
- the present invention also makes it possible to highlight the genes involved in the pigmentation of the skin, the hair and the integuments, within the three chromosomal zones of the invention.
- a particular use envisaged by the present invention therefore consists in using SNPs markers within each of the chromosomal regions of interest with the aim of localizing more finely the genes involved in pigmentation, and more particularly those involved in the progressive interruption or sudden pigmentation of the skin or integuments.
- the present invention is based on the identification by the inventors of genes, on chromosomes 3, 5 and 11 human, involved in the phenomenon of pigmentation or depigmentation. This genetic basis enabled them to envisage the uses in therapy and in cosmetics described; previously, as well as the diagnostic methods, illustrated above.
- the particularly preferred uses are those which take advantage of the combination of the results obtained for the three chromosomal zones of the invention.
- a first combination use in the field of cosmetics and therapy preferably makes use of at least two polynucleotide fragments, the sequence of each of which corresponds, at least in part, to that of one of the 5 genes of the invention of the human chromosome 3, or of that of one of the genes of the invention of the human chromosome 5, or of that of one of the 18 genes of the invention of the human chromosome 11.
- use is therefore made of two or more fragments, the sequence of at least one corresponding, at least in part, to those previously mentioned on chromosomes 3, 5 and 11, the sequence of the other or others corresponding, at least in part, either to those previously mentioned on chromosomes 3, 5 and 11, or to that of a gene from human chromosome 6 chosen from the genes HLAG, NT_007592.445, NT_.007592.446, NT_007592.
- Preferred genes are DDX31 and GTF3C4.
- At least two have sequences corresponding to two distinct chromosomes, or to two distinct genes. It is also conceivable that for the same gene, the different fragments which are used have different chemical natures, for example DNA for the first fragment and RNA for the second. It is also conceivable that different fragments have a sequence corresponding to the same gene, but with the slight variations permitted by the definition of “correspondence sequences”, that is to say at most one different nucleotide in 10, preferably 1 in 100
- the fragments contain at least 18 successive nucleotides, these 18 nucleotides forming the sequence which must correspond at least partially to one of the genes mentioned.
- One of the fragments contains at least 18 successive nucleotides corresponding at least partially to one of the genes of the invention.
- these two fragments are preferably carried by separate molecules. It is also conceivable that these two fragments are, for example, part of the same vector. According to a preferred case, the different fragments are of the same chemical nature, for example DNA for all the fragments.
- the polynucleotide fragments are used in the manufacture of a medicament.
- a second use of combination envisaged by the present invention, in the field of therapy and in that of cosmetics, is the use of a combination of at least two agents each modulating the function of a selected DNA fragment from the fragments belonging to and / or corresponding to all or part of one of the 5 genes of the invention of human chromosome 3, or of one of the genes of the invention of human chromosome 5, or of one of 18 genes for the invention of human chromosome 11.
- a preferred use takes advantage of the results obtained on chromosomes 6 and 9. It is therefore advantageously made use of two or more agents, one at least modulating the function of a DNA fragment chosen from the fragments belonging and / or corresponding to all or part of the genes previously mentioned on chromosomes 3, 5 and 11, the other agent (s) each modulating the function of a DNA fragment chosen from the fragments belonging to and / or corresponding to all or part of a gene of the invention or a gene chosen from the genes HLAG, NT_007592.445, .NT_007592.446, NT_007592.506, NT_007592.507, NT_007592.508, HSPAIB, G8, NEUl, NG22, BAT8, HLA-DMB , HLA-DMAj BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638, NT_007592.459, NT_00759
- Preferred genes are the DDX31 and GTF3C4 genes.
- at least two modulate the function of DNA fragments corresponding to two distinct chromosomes, or two distinct genes. It is also conceivable that for the same chromosomal zone, the different agents which are used modulate different functions of the same DNA fragment.
- the DNA fragments whose function is modulated according to the invention preferably contain at least 18 successive nucleotides, these 18 nucleotides forming the sequence which must correspond at least partially to one of the genes mentioned.
- One of the fragments contains at least 18 successive nucleotides corresponding at least partially to one of the genes of the invention. All the preferred uses of such agents have already been explained in detail in the section describing the second use according to the invention.
- a third combination use envisaged by the present invention is the use of a combination of at least two agents each modulating the function of the expression product of a DNA fragment chosen from the fragments belonging to and / or corresponding to all or part of one of the 5 genes of the invention of human chromosome 3, or of one of the genes of invention of human chromosome 5, or alternatively d one of the 18 genes of the invention of human chromosome 11.
- a preferred use takes advantage of the results obtained on chromosomes 6 and 9. It is therefore advantageously made use of two or more agents, one at least modulating the function of the expression product of a DNA fragment chosen from fragments belonging to and / or corresponding to all or part of the genes; of the invention on chromosomes 3, 5 and 11, the other agent (s) each modulating the function of the expression product of a chosen DNA fragment among, the fragments belonging to and / or corresponding to all or part of the previously mentioned genes, or else to all or part of a gene chosen from the genes HLAG, NT_007592.445, NT_007592.446, NT_007592.506, NT_007592.507, NT_007592 .508, HSPAIB, G8, NEUl, NG22, BAT8, HLA-DMB, HLA-DMA, BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638,
- n592.459 007592.459, NT_007592.457, FREQ, NT_030046.18, NT_030046.17, GTF3C5, CEL, CELL, FS, ABO, BARLH1, DDX31, GTF3C4 and Q96MA6.
- Preferred genes are the DDX31 and GTF3C4 genes.
- the different agents which are used modulate different functions of the same product of expression of a DNA fragment, or else modulate different products of expression of a DNA fragment, for example RNAs at different stages of maturation, or else RNAs from different splices.
- the DNA fragments whose function of the expression product is modulated according to the invention preferably contain at least 18 successive nucleotides, these 18 nucleotides forming the sequence which must correspond at least partially to one of the genes mentioned.
- One of the fragments contains at least 18 successive nucleotides corresponding at least partially to one of the genes of the invention. All the preferred uses of such agents have already been explained in detail in the section describing the third use according to the invention.
- a fourth combination use envisaged by the present invention is the use of a combination of at least two expression products of DNA fragments chosen from the fragments belonging to and / or corresponding to all or part of one of the 5 genes of the invention of human chromosome 3, or of one of the genes of the invention of human chromosome 5, or of one of the 18 genes of l invention of human chromosome 11.
- a preferred use takes advantage of the results obtained on chromosomes 6 and 9. It is therefore advantageously made use of two or more expression products, at least one of which is the expression product of a DNA fragment chosen from the fragments belonging to and / or corresponding to all or part of one of the genes of the invention, the others being each the product of expression of a DNA fragment chosen from the fragments belonging and / or corresponding to any or part of a gene of the invention or of a gene chosen from the genes HLAG, NT_007592.445, NT_007592.446, NT_007592.506, NT_007592.507, NT_007592.508, HSPAIB, G8, NEUl, NG22, BAT8 , HLA-DMB, HLA-DMA, BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638, NT_007592.459, NT_007592.457, FR
- the various expression products which are used are derived from DNA fragments corresponding to two distinct chromosomes, or to two distinct genes. It is also possible to envisage that for the same chromosomal area of the invention, the different expression products which are used are derived from the same DNA fragment, it may for example be RNA at different stages of maturation, or RNA from different splices. The same possibilities are offered for the polypeptides resulting from the translation of RNAs.
- the DNA fragments whose expression products are used according to the invention preferably contain at least 18 successive nucleotides, these 18 nucleotides forming the sequence which must correspond at least partially to one of the genes mentioned.
- One of the fragments contains at least 18 successive nucleotides corresponding at least partially to one of the genes of the invention.
- the expression products are used in the manufacture of a medicament.
- the cosmetic uses are preferably in the field of pigmentation.
- a very interesting combination comprises at least one polynucleotide fragment or an expression product of a sequence corresponding to all or part of the DDX31 or GTF3C4 gene of the human chromosome 9. . ; ⁇ • ,
- the present invention also relates to combination methods. These methods are used to determine a possible predisposition to premature canities.
- a combination method for determining a predisposition to premature canities includes a first step of selecting at least two markers which will be used in the following steps.
- the markers selected are chosen from markers belonging to the region of human chromosome 3 comprising the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097, the markers belonging to the region of human chromosome 5 comprising the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETFl, HSPA9B, PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and FLJ1115 region markers 11 human including the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF
- markers belong to one of these genes.
- at least one of the markers selected is chosen from the markers belonging to one of the regions previously mentioned on chromosomes 3, 5 and 11, the other marker or markers selected belonging to the chromosomal regions previously mentioned, or that of chromosome 6 human between the markers D6S1629 and D6S257, or that of human chromosome 9 between the marker D9S290 and the telomeric region (telomer of the long arm).
- the markers are preferably within a gene chosen from the genes HLAG, NT_ 007592.445, NT_007592.446, NT_007592.506, NT_007592.507, NT_007592.508, HSPAIB, G8, NEUl, NG22 , BAT8, HLA-DMB, HLA-DMA, BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638, NT_007592.459, NT_007592.457, FREQ, NT_030046.18, NT_030046.17, GTF3C5 , CEL, CELL, FS, ABO, BARLHl, DDX31, GTF3C4 and Q96MA6.
- Preferred genes are the DDX31 and GTF3C4 genes.
- at least two do not belong to the same
- the next step in implementing a method according to the invention consists, for the markers chosen, in determining the alleles present> in a sample of genetic material derived from; the individual undergoing the diagnostic test? Of them. different alleles, carried by the two versions of the chromosome, can be identified.
- the combinations of the invention can be incorporated into a cosmetic or pharmaceutical composition as described above.
- a method of combining the invention may include the additional step of comparing the allaque form of the selected markers with the allaque form of these same markers in other individuals.
- This additional comparison step may be necessary in order to establish the diagnosis.
- it may be useful to make a comparison with the shape of the markers in individuals known to have premature canities and possibly also with the shape of the markers in individuals known to be free from such a predisposition.
- a particularly interesting situation consists in comparing the alleles of markers selected for alleles of these same markers in other individuals of the same family as the individual to be diagnosed.
- the present invention finally makes it possible to highlight the genes involved in the pigmentation of the skin, hair and integuments, within the five chromosomal zones mentioned.
- a particular use envisaged by the present invention therefore consists in using a combination of the SNPs markers within each of the chromosomal regions of interest with the aim of localizing more finely the genes involved in pigmentation, and more particularly those involved in the interruption. progressive or sudden pigmentation of the skin or integuments.
- the present invention relates to a kit comprising a combination of at least two polynucleotide fragments chosen from those comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of one of the 5 genes of the invention of human chromosome 3, or one of the genes of the invention of human chromosome 5,. or one of the 18 genes of the invention of human chromosome 11.
- the kit comprises two or more fragments, the sequence of at least one corresponding, at least in part, to those previously mentioned on chromosomes 3, 5 and 11, the sequence of the other or of each of the others corresponding, at least in part, or to those previously mentioned, or else to that of a gene chosen from the genes HLAG, NT_007592.445, NT_007592.446, NT_007592.506, NT_007592.507, NT_007592.508, HSPAIB, G8, NEUl, NG22, BAT8, HLA-DMB, HLA-DMA, BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638, NT_007592.459, NT_007592.457, FREQ, NT_030046.
- NT_030046.17 GTF3C5, CEL, CELL, FS, ABO, BARLHl, DDX31, GTF3C4 and Q96MA6.
- Preferred genes are the DDX31 and GTF3C4 genes.
- Figure 1 Composition of families analyzed for linkage with the candidate region
- Figure 2 Candidate region for PC on chromosome 3: Chromosomal location and distribution of markers
- Figure 3 Graphical representation of the NPL scores obtained for the non-parametric multi-point global genome linkage analysis on the CP families for the chromosomes selected.
- FIG.A Chromosomes 3 and 5;
- Figure 3B Chromosomes 6 and 9
- FIG. 3C Chromosomel 1
- Figure 4 Schematic representation of the CP loci identified on chromosomes 11,
- FIG. 4A Chromosome 11, locus 11 ql4-q22
- Figure 4B Chromosome 5, locus 5q31-q32 - ”.
- FIG. 4C Chromosome 3, locus 3pl4.1-pl2.3
- FIG. 4D Chromosome 6, locus6p21 -p 12
- Figure 4E Chromosome 9, locus 9q34
- the columns display the average Lod score, the standard deviation, the minimum Lod score, the maximum Lod score and the group (A-E) in which the family places themselves according to their score.
- Figure 6 Potential Lod scores by family as a function of the rate of genetic heterogeneity in PC.
- Figure 7 Lod scores detailed by family as a function of the rate of genetic heterogeneity of the PC: new simulation after collection of the new families.
- Figure 8 New simulation on the finalized families for the search for candidate chromosomal regions. Lod potential scores depending on the rate of heterogeneity.
- Figure 10 Comparison of the composition of families between the candidate-region analysis and the Genome-global analysis.
- Figure 11 Potential Lod scores as a function of the rate of heterogeneity of the PC. The results are expressed by family.
- Figure 12 Probability (in%) of reaching or exceeding a Lod score of 1, 2 or 3 for each rate of heterogeneity. The results are expressed by family.
- Figure 13 Composition of the 4 pools.
- the AI and Ail pools are made up of individuals with premature canities.
- the two control pools BI and BH are composed of “crossed” individuals for origin and age with individuals suffering from premature canities.
- PC premature canities
- A- Epoch 1 Determination of the potential of the study. A first selection of the most informative families is carried out by a simulation of link analysis.
- B- Epoch 2 Medical confirmation of phenotypes and collection of blood samples from preselected families. This verification campaign results in a new list of families applying for the study. A new simulation of connection makes it possible to estimate the potential of F corrected sample.
- C- Epoch 3 Analysis of genetic link with the candidate chromosomal region for PC. First phase of DNA analysis for the chromosomal region which could contain the CP genes.
- D- Epoch 4 Analysis of global genetic linkage of PC on the whole human genome. Analysis of family segregations on the DNA of the 22 autosomal chromosomes and the X chromosome in order to detect the regions that bind to the CP trait.
- A- Epoch 1 Choice of families with the help of link analysis simulation
- Twenty-nine families among the 65 were selected, based on structural criteria (total number of individuals, affected, available), to be analyzed in simulation.
- a) software generates a series of replicates of the code file by assigning simulated genotypes. The file obtained for each family explores several allelic combinations (genotypes) in each of the individuals, b) software then analyzes each of the replicates to estimate the possible Lod scores (Z) for genetic link analysis in each family.
- the results, in the form of minimum, average and maximum Lod scores thus make it possible to assess the potential of each family in this type of study. Obviously, these estimates only remain valid in the case where each individual has been assigned a correct phenotype.
- the phenotype In case of uncertainty, the phenotype should be reported as unknown; it is therefore not taken into account in the study and therefore does not need to be sampled.
- the Lod score decreases (in variable proportions) each time an individual is removed for an uncertain phenotype.
- the family trees have been redesigned using pedigree management software, which also builds coded files (prephnk files) for genetic linkage analysis.
- coded files prephnk files
- an availability code (cd) (table 1) is assigned. It therefore weighs, by a code of 0 to 3, the informative nature of each individual in the study.
- the following criteria have been defined for assigning the phenotype according to age in individuals under 30 years of age. However, during the final clinical examination, it is desirable that the definition of the phenotype be more quantitative.
- the number of replications was finally fixed at 200. • •, The frequency of the trait a was set at 1%. The number of possible alleles for the genotype is fixed at 6 with an equivalent frequency for each.
- Table 3 gives an indication of the potential of the GENORMAX families as a function of the maximum Lod score (Zmax) achieved (group A-E).
- Figure 5 reports the simulated Lod score for each family.
- Table 3 Family statistics / maximum Lod scores. The detailed results are shown in the table in Figure 5.
- the maximum Lod score can only be reached when a DNA marker is 100% informative in a family. Most often, even with the type of markers used (the most informative markers in the chromosomal regions to be visited), the Lod score will not reach its maximum value.
- Table 4 Lod score obtained for a series of distances from the marker to the trait locus (Lod score maximum in bold).
- the families of group A will be informative in a genetic linkage analysis for the localization of the genes of premature canities. For this, no uncertainty should remain regarding the phenotype. When in doubt, it is advisable to exclude individuals or even families from the study. However, in some of these families, the high proportion of affected / unaffected individuals (sometimes all affected children) should encourage extreme distrust of the genetic trait at 100% of the trait. Of course, it is not excluded that in certain families, the CP gene was transmitted simultaneously by the paternal and maternal branches of the first generation. In this case, the small children would all be affected (Can28, 43, 53 ). In order to be able to positively consider these few families, it is highly desirable to verify this hypothesis.
- group B The families in group B are themselves very interesting because they allow the sample to be enlarged, even if individually they will not be able to access a significant Lod score in most cases. In groups, they can however consolidate the Lod score, especially if it turns out that the trait was genetically heterogeneous (slightly).
- the families of group C can also be used in studies of replication of the results of genetic linkage.
- the families of groups D and E (Zmax ⁇ 2) are not very informative for a genetic hedge analysis.
- B- Period 2 Collection of samples, confirmation of phenotypes and new estimates of the study's potential
- This phenotype medical verification campaign helped to confirm many of the PC diagnoses, but not all as expected.
- Table 5 presents the results obtained for all 8 families in 3 situations of genetic heterogeneity (0%, i.e. all families linked to the same locus, 50% or only half of the families linked to the locus, 70% or only about a third of families linked).
- Table 7 New simulation after collection of new families. The potential Lod scores are expressed as a function of the rate of genetic heterogeneity.
- Heterogeneity rate 0% 50% 70% Lod score 1. 000 99,500 57,000 27,000 2. 000 95,500 31,000 8,500 3. 000 84,500 14,000 3,500
- the region 3pl4.1-pl2.3 is called the “candidate region” (CR) because it is associated with premature canities through Klein Waardenburg disease (type IIA) with which the trait is associated.
- Table 9 New simulation on families finalized for research on the candidate chromosomal region. Lod potential scores based on the rate of genetic heterogeneity. The detailed results are shown in the table in Figure 8.
- Table 10 Probabilities (in%) of reaching or exceeding Lod score of 1, 2 or 3 for each rate of heterogeneity. The detailed results are shown in the table in Figure 9.
- NPL non-parametric
- CAN53 shows a relatively high hedge for this chromosomal region.
- D- Epoch 4 Analysis of the global genetic link between PC and the genome.
- Table 12 Comparison of the number of individuals studied in each family between the candidate-region analysis and the genome-global analysis, (detailed composition of families, table in Figure 10)
- the probability of finding a significant link (Table 14 and Figure 12) with 4/5 of families living at the same locus is 50%. This result is based on an average lod score which is conservative.
- Table 14 Probabilities (in%) of reaching or exceeding Lod score of 1, 2 or 3 for each rate of heterogeneity
- Analyzes can therefore indicate significant links if the heterogeneity of the sample does not exceed 20% (i.e. only 1/5 of families not binding to a single major locus), but it is still possible to identify a link in the case where half of the families are not linked to this locus.
- the DNA of 96 individuals belonging to the 12 families selected was genotype for 400 polymorphic markers distributed on the 22 autosomes and the X chromosome (Table 15) according to an average inter-archer interval of 9.2 cM (density). These are DNA microsatellites, which are composed of tandem repeats of the dinucleotide type (CA) facedfrom the Généthon collection (Evry, France).
- CA dinucleotide type
- Table 15 Number of markers analyzed for each chromosome during the wide scan genome
- the average heterozygosity rate observed is 0.70, and the average size of the inter-marker interval is 9.2 cM.
- the inventors have carried out several types of analysis in order to optimize their performance in order to find a link between a region of the genome and the CP.
- Parametric analysis more efficient in terms of power, takes into account the mode of transmission of the trait and is most suitable in the case of monogenic traits.
- the non-parametric analysis which makes it possible to identify a link even if the mode of transmission supposed is erroneous, is also more robust in the case of multigem traits.
- the inventors used the methods already mentioned, the 2-point method (iterative analysis between the line and each marker) and the multipoint method (global analysis on each chromosome according to a map of markers).
- NPL non-parametric
- Figure 3 reports the NPL scores obtained for the analysis of non-parametric hedge on chromosomes 3, 5, 11, 6 and 9.
- Tables 16 (best 2-point analysis results) and 17 (best multipoint analysis results) report the best results obtained for the chromosomes selected.
- the NPL score is moreover in the range of suggestivity in a case of monogenism or in a case of multigenism (suggestivity: monogenism 2 ⁇ Z-all ⁇ 3; multigenism 2.2 ⁇ Z-all ⁇ 3.6).
- the inventors identify a region between positions 100 and 115 (between Dl 1S898 and Dl 1S925) ( Figure 4A).
- This region collects the best PL and bipoint result of these analyzes which is located at a recombination distance (theta) 0.14 (approximately 14 cM) from the marker D5S422.
- a recombination distance 0.14 (approximately 14 cM) from the marker D5S422.
- Example 1 Following the work presented in Example 1, the inventors continued the analysis of the regions of chromosomes 3, 5 and 11 using SNP-based technology, in order to highlight the genes involved in canities. early.
- GG global genome MP: multipoint
- SNPs single nucleotide polymorphism
- SNPs single nucleotide polymorphism
- the number of SNPs is estimated to be around 0.8 SNPs per 1000 nucleotides (coding and non-coding sequences combined), which makes it possible to establish a true mapping of the human genome using SNPs.
- SNPs are often classified into different categories, in particular according to whether they are in a coding region or not, in a regulatory region or in another non-coding region of the genome, whether the polymorphism modifies the coded amino acid or not, etc. .
- SNPs are now better known and referenced, as well as their position in the genome (GDB).
- GDB Human Genome Project program
- Different methods have been developed to demonstrate these polymo ⁇ hisms between different individuals, often based on the methods used to detect point mutations (RFLP-PCR, hybridization with allele specific oligomers, mini-sequencing, direct sequencing ).
- RFLP-PCR point mutations
- mini-sequencing mini-sequencing
- direct sequencing a point mutations
- MALDI-TOF technology matrix-assisted laser deso ⁇ tion / ionization time-of-flight mass spectiometry
- the inventors defined very precisely the regions of chromosomes 3, 5 and 11 to be analyzed with SNPs.
- 3848 SNPs belonging to the previous regions were pre-selected on certain criteria (SNPs candidates in silico) and 3227 were selected following an experimental validation step.
- the inventors assembled the DNAs of the different individuals with premature canities and of the 'control' individuals into different groups, then genotyped these different groups using 1264 SNPs chosen from among 3227. The different steps are described more fully in the following sections.
- the inventors defined more precisely the regions of interest on chromosomes 3, 5 and 11, from the results obtained thanks to the analysis with microsatellite markers (see the work described in Example 1) of the 12 families selected (see Table 6).
- region C The region on chromosome 3, called region C, is defined by its chromosomal position as well as by three other types of coordinates for optimal precision and security in the definition of this region for the subsequent steps. The same is true for the region of chromosome 5, called region D, and for the region of chromosome 11, called region E.
- the following table lists the different cartographic characteristics of regions C, D and E (code and size of the region in Mb and Kb, number of microsatellite markers that bound the regions, position in the genome sequence, number of an SNP).
- the two columns for the positions on the genome differ in the different version of database (NCBI UCSC freeze April 2002 and June 2002).
- the cumulative size of the three C + D + E regions is 77,351 Kb, or 77,106 bp.
- the position of the sequence is expressed as a function of the version number of the human genome database according to a UCSC update (http: //genome.cse.ucsc. edu /) and / or a human genome sequence assembly version number, in April 2002 (either NCBI Build29) or June 2002 (either NCBI Build30).
- 3332 SNPs were preselected in particular by removing the SNPs distant by less than 14kb.
- the 3332 SNPs selected were analyzed on 92 control individuals (individuals from the Center for the Study of Human Polymohism) in order to validate the presence of at least two alleles for each of the SNPs (validation of polymohismism).
- the DNAs of the different individuals with the trait 'early canities' (CP) and that of the control individuals were assembled. This assembly was carried out so that each of the DNAs is represented in an equimolar manner, in order to guarantee that no individual has a preponderant influence on the result compared to another. To this end, the exact concentration of each of the DNAs was measured by the “Picogreen” method in the various samples of the individuals.
- Groups were formed taking into account a “phenotypic intensity of canity score” which was assigned for each individual as follows.
- AI group this group is made up of the DNA of 72 PC individuals whose phenotypic score is 4 or 5.
- AU group this group is made up of DNA from 132 PC individuals, whose phenotypic score is 2, 3, 4 or 5.
- BI and BII groups are made up of the DNA of control individuals whose geographic origin is close to that of CP individuals. For these control individuals, the selection criteria were: (i) an age greater than 40 years, (ii) the absence of sign of canities in the control individual, (iii) the absence of a family concept of canities.
- the criteria for matching with an individual from the AI or AU group are identical geographic origin, same sex and identical hair color at 18 years of age.
- each CP individual in the AI group is represented by a control individual in the BI group whose place of geographic origin is close or identical. The same is true for each individual in the AH group.
- the rigor of matching according to the rules set by the inventors is the guarantee of the relevance of statistical analyzes comparing genomic data from these individuals whether they are grouped in pools or compared individually.
- a battery of 2142 SNPs from groups 1 and 2 were thus validated for a minimum frequency of the rarest Pallele of 0%.
- Some SNPs of groups 3 and 4 (frequency of the rare allele ⁇ 10%) were also selected in order to be able to complete certain zones where there would not be enough SNPs from groups 1 and 2.
- 3227 SNPs were thus validated.
- 1,264 were chosen during a new selection stage. This new selection is based on the following criteria:
- the next step was to determine their allelotype, that is to say the frequency of each of the alleles, for the 4 groups of DNA assembled (pooled) depending on the severity and precocity of the phenotype (see the definition of the 4 groups in step 3 and Figure 13).
- the Alabank frequency of the two alleles was determined for each SNP in the 4 groups.
- allelotype is determined by this method, and not the genotype.
- the statistical significance of the allelic frequency differences between the groups AI and BI or AU and BII is estimated by the value "p" representing the significance. The lower the p-value, the more statistically significant the difference.
- -CHROM chromosome number
- -AI_A2 is the frequency of allele 2 (the higher mass allele in MALDI-TOF in the AI pool)
- -BI_A2 is the frequency of allele 2 (the higher mass allele in MALDI-TOF in the pool BI)
- -DAI-BI is the difference in frequency of allele 2 between pool AI and pool BI
- -P-value_I is the p-value calculated for the comparison of the frequencies of the AI and BI pools.
- -P ⁇ 0.05: value 0 when the p-value is> 0.05; value 1 when the p-value is ⁇ 0.05 -Code: code (ind, dbs, 2, 3), depending on the nature of the result (see table of codes), indicating whether it is an individual positive SNP, or distributed in double spot or cluster of 2, 3 or more.
- this criterion indicates whether the frequencies of 2 or 3 positive cluster SNPs (contiguous positive SNPs) are compatible with the existence of a haplotype. To do this, the frequencies of the 2 alleles (positive SNPs in the cluster), frequent allele and rare allele (lower and upper allele), are statistically analyzed to determine if there is a linkage disequilibrium (LD Linkage desequilibrium) resulting in significant p-value.
- LD Linkage desequilibrium linkage disequilibrium
- Linkage disequilibrium is a situation where 2 genes (alleles) segregate together at a higher frequency than the frequency predicted by the product of their frequencies individual. This means that the two genes are not independent since they segregate together more frequently than expected by the statistics, so there is a lack of independence between alleles located close to each other on the same chromosome. In addition, this takes into account the distance, and therefore SNPs distant from more than 100-110 Kbs are eliminated.
- i- region C Region C is characterized by a relatively large number of isolated positive SNPs. On the other hand, the number of double spots or clusters is relatively low. LD analysis
- -CCK gas ⁇ rin / cholecystokinintypeb receptor (cck-b receptor)
- C2 f52846896-5291394n: -CACNA1D: voltage-dependent 1-type calcium channel alpha-ld subunit
- Region D is characterized by a relative density in clusters and the LD and freq analysis also shows several regions of interest. Seven zones (D1, D2, D3, D4, D5, D6, D7) are noted for their cluster arrangement, their LD potential and the arrangement of the allelic frequencies. Among these 7 zones, 5 (in bold; D2, D3, D5, D6, D7) seem to be more promising due to the significance of the comparison of at least one SNP.
- -HNRPAO heterogeneous nuclear ribonucleoprotein aO (hnrnp aO).
- -CDC25C map / microtubule affinity-regulating kinase 3 (ec 2.7.1.27)
- EGR1 early gro th response protein 1 (egr-1) (krox-24 protein)
- -ETFl eukaryotic peptide chain release factor subunit 1 (erfl)
- HSPA9B stress-70 protein, mitochondrial precursor
- -PDGFRB beta platelet-derived growth factor receptor precursor (ec 2.7.1.112)
- -TCOFl Treacle Protein (Treacher Collins Syndrome Protein).
- -ALI 33039 predicted
- -CD74 hla class ii histocompatibility antigen, gamma chain
- -G3BP ras-gtpase-activating protein binding protein 2
- GLRAl glycine receptor alpha-1 chain precursor D7 (153463449-153854650)
- C5orf3 predicted
- -GALNTIO putative udp-galnac: n-acetylgalactosaminyltransferase polypeptide
- Region E is also characterized by a relative density in clusters and the LD and freq analysis shows 6 regions of interest (E1, E2, E3, E4, E5, E6). Three zones (in bold, E2,
- -GUCY1A2 soluble guanylate cyclase, alpha-2 chain (ec 4.6.1.2) E2 (110056711-110546142)
- vasopressin-activated calcium-mobilizing receptor (vacm-1) (cullin homolog 5)
- -ACAT1 acetyl-coa acetyltransferase, mitochondrial precursor (ec 2.3.1.9)
- -NPAT muclear protein ataxia-telangiectasia locus; el4 gene;
- IGSF4 immunoglobulin superfamily, member 4; nectin-like protein 2 E5 (118532530-118685957)
- transgelin smooth muscle protein 22-al ⁇ ha
- sm22-al ⁇ ha ws3-10
- 22 kda actin-binding protein 22 kda actin-binding protein
- This lotion is applied daily to the areas to be treated and preferably to the entire scalp for at least 10 days and preferably 1 to 2 months.
- This shampoo is used for each wash with an exposure time of approximately one minute. Prolonged use, of the order of two months, leads to the gradual repigmentation of gray hair. This shampoo can also be used as a preventive measure to delay bleaching of the hair.
- This gel is applied to the areas to be treated twice a day (morning and evening) with a terminal massage. After three months of application, a repigmentation of the hairs or hair of the treated area is observed.
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Abstract
Description
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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EP03748204A EP1520042A2 (fr) | 2002-07-10 | 2003-07-09 | Genes des chromosomes 3,5 et 11 impliques dans la canitie precoce |
CA002491235A CA2491235A1 (fr) | 2002-07-10 | 2003-07-09 | Genes des chromosomes 3,5 et 11 impliques dans la canitie precoce |
JP2004520753A JP2005532407A (ja) | 2002-07-10 | 2003-07-09 | 若白髪に関与する染色体3、5、11の遺伝子 |
BR0305436-5A BR0305436A (pt) | 2002-07-10 | 2003-07-09 | Utilização de pelo menos um fragmento polinucleotìdico e de uma combinação de pelo menos dois fragmentos poliniucleotìdicos, processo para o diagnóstico de uma predisposição a encanecimento precoce em um indivìduo, e, kit |
AU2003267513A AU2003267513A1 (en) | 2002-07-10 | 2003-07-09 | Chromosome 3, 5 and 11 genes involved in premature canities |
US11/031,033 US20050208010A1 (en) | 2002-07-10 | 2005-01-10 | Genes from Chromosome 3, 5 and 11 involved in premature canities |
US11/583,512 US20070065389A1 (en) | 2002-07-10 | 2006-10-18 | Genes from chromosomes 3, 5 and 11 involved in premature canities |
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FR02/08697 | 2002-07-10 | ||
FR0208697A FR2842105B1 (fr) | 2002-07-10 | 2002-07-10 | Localisation des genes impliques dans la canitie precoce |
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US11/031,033 Continuation US20050208010A1 (en) | 2002-07-10 | 2005-01-10 | Genes from Chromosome 3, 5 and 11 involved in premature canities |
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WO2004007764A3 WO2004007764A3 (fr) | 2004-04-08 |
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PCT/FR2003/002153 WO2004007764A2 (fr) | 2002-07-10 | 2003-07-09 | Genes des chromosomes 3,5 et 11 impliques dans la canitie precoce |
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EP (1) | EP1520042A2 (fr) |
JP (1) | JP2005532407A (fr) |
AU (1) | AU2003267513A1 (fr) |
BR (1) | BR0305436A (fr) |
CA (1) | CA2491235A1 (fr) |
FR (1) | FR2842105B1 (fr) |
WO (1) | WO2004007764A2 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008058143A2 (fr) | 2006-11-06 | 2008-05-15 | Qualcomm Incorporated | Procédés et appareil d'attribution de puissance et/ou de sélection de débit pour des opérations ul mimo/simo avec des considérations par |
EP2113286A1 (fr) | 2008-04-30 | 2009-11-04 | L'Oreal | Utilisations de BNIPXL-beta dans la canitie précoce |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2864899A1 (fr) * | 2004-01-08 | 2005-07-15 | Oreal | Differents genes impliques dans la canitie precoce |
WO2005068650A2 (fr) * | 2004-01-08 | 2005-07-28 | L'oreal | Divers genes impliques dans des canities precoces |
JP5695384B2 (ja) | 2009-10-05 | 2015-04-01 | 花王株式会社 | 毛髪形状感受性遺伝子 |
JP5695383B2 (ja) | 2009-10-05 | 2015-04-01 | 花王株式会社 | 毛髪形状感受性遺伝子 |
JP5695385B2 (ja) | 2009-10-05 | 2015-04-01 | 花王株式会社 | 毛髪形状感受性遺伝子 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995001773A1 (fr) * | 1993-07-07 | 1995-01-19 | Trustees Of Boston University | Stimulation du bronzage par des fragments d'adn |
WO1996036691A1 (fr) * | 1995-05-16 | 1996-11-21 | Ramot-Univ. Authority For Applied Research And Industrial Development Ltd. | Gene de l'ataxia-telangiectasie et son organisation genomique |
WO2000047770A1 (fr) * | 1999-02-15 | 2000-08-17 | Catalyst Biomedica Ltd. | Locus de susceptibilite de l'osteoarthrite |
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2002
- 2002-07-10 FR FR0208697A patent/FR2842105B1/fr not_active Expired - Fee Related
-
2003
- 2003-07-09 EP EP03748204A patent/EP1520042A2/fr not_active Withdrawn
- 2003-07-09 AU AU2003267513A patent/AU2003267513A1/en not_active Abandoned
- 2003-07-09 CA CA002491235A patent/CA2491235A1/fr not_active Abandoned
- 2003-07-09 WO PCT/FR2003/002153 patent/WO2004007764A2/fr not_active Application Discontinuation
- 2003-07-09 JP JP2004520753A patent/JP2005532407A/ja active Pending
- 2003-07-09 BR BR0305436-5A patent/BR0305436A/pt not_active IP Right Cessation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995001773A1 (fr) * | 1993-07-07 | 1995-01-19 | Trustees Of Boston University | Stimulation du bronzage par des fragments d'adn |
WO1996036691A1 (fr) * | 1995-05-16 | 1996-11-21 | Ramot-Univ. Authority For Applied Research And Industrial Development Ltd. | Gene de l'ataxia-telangiectasie et son organisation genomique |
WO2000047770A1 (fr) * | 1999-02-15 | 2000-08-17 | Catalyst Biomedica Ltd. | Locus de susceptibilite de l'osteoarthrite |
Non-Patent Citations (4)
Title |
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BARSH G S: "The genetics of pigmentation: from fancy genes to complex traits" TRENDS IN GENETICS, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 12, no. 8, 1 août 1996 (1996-08-01), pages 299-305, XP004037138 ISSN: 0168-9525 * |
HEMMINKI A ET AL: "Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis" NATURE GENETICS, NEW YORK, NY, US, vol. 15, no. 1, 15 janvier 1997 (1997-01-15), pages 87-90, XP001105579 ISSN: 1061-4036 * |
OETTING W S ET AL: "Molecular basis of albinism: mutations and polymorphisms of pigmentation genes associated with albinism." HUMAN MUTATION. UNITED STATES 1999, vol. 13, no. 2, 1999, pages 99-115, XP008018509 ISSN: 1059-7794 * |
TASSABEHJI M ET AL: "Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene" NATURE GENETICS, NEW YORK, NY, US, vol. 8, no. 3, novembre 1994 (1994-11), pages 251-255, XP000909097 ISSN: 1061-4036 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008058143A2 (fr) | 2006-11-06 | 2008-05-15 | Qualcomm Incorporated | Procédés et appareil d'attribution de puissance et/ou de sélection de débit pour des opérations ul mimo/simo avec des considérations par |
EP2113286A1 (fr) | 2008-04-30 | 2009-11-04 | L'Oreal | Utilisations de BNIPXL-beta dans la canitie précoce |
Also Published As
Publication number | Publication date |
---|---|
BR0305436A (pt) | 2004-09-28 |
AU2003267513A8 (en) | 2004-02-02 |
FR2842105A1 (fr) | 2004-01-16 |
FR2842105B1 (fr) | 2012-12-28 |
WO2004007764A3 (fr) | 2004-04-08 |
AU2003267513A1 (en) | 2004-02-02 |
CA2491235A1 (fr) | 2004-01-22 |
EP1520042A2 (fr) | 2005-04-06 |
JP2005532407A (ja) | 2005-10-27 |
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