EP1520042A2

EP1520042A2 - Chromosome 3, 5 and 11 genes involved in premature canities

Info

Publication number: EP1520042A2
Application number: EP03748204A
Authority: EP
Inventors: Olivier De Lacharriere; Jean-Louis Blouin; Claire Deloche; Stylianos Antonarakis
Original assignee: LOreal SA
Current assignee: LOreal SA
Priority date: 2002-07-10
Filing date: 2003-07-09
Publication date: 2005-04-06
Also published as: WO2004007764A2; WO2004007764A3; FR2842105B1; JP2005532407A; FR2842105A1; AU2003267513A1; CA2491235A1; BR0305436A; AU2003267513A8

Abstract

The invention concerns the use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whereof the sequence corresponds to all or part of a human chromosome 3 gene selected among KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097 or whereof the sequence corresponds to all or part of a human chromosome 5 gene selected among KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETF1, HSPA9B, PCDHAI to PCDHA13, CSF1R, RPL7, PDGFRB, TCOF1, AL133039, CD74, RPS14, NDST1, G3BP, GLRA1, C5orf3, MFAP3, GALNTIO and FLJ11715 genes, or whereof the sequence corresponds to all or part of a human chromosome 11 gene selected among GUCYIA2, CULS, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650 genes, the use of agents capable of modifying the function attached to one said regions, the use of expression products of one of said regions and the use of agents capable of modifying the function of said expression products, for cosmetic, therapeutic or diagnostic applications.

Description

GENES OF CHROMOSOMES 3, 5 and 11 INVOLVED IN THE

EARLY CANITIE

Canceling or minimizing the effects of aging as much as possible is a growing concern. In this context, removing white hair deemed unsightly with coloring treatment shampoos is now a very common activity. It is clear, however, that even if this technique effectively cancels the effects of the phenomenon, it does not affect the causes. Therefore, this solution is temporary and must be renewed frequently. In this context, the inventors chose to explore the appearance of white hair, or canities, from a totally new angle, that of genetics.

Indeed, exploring canities through its genetic aspect makes it possible to highlight the deep mechanisms of depigmentation. This also helps identify the genes that are involved in canities. This identification opens the door to many applications, in the field of hair care, both cosmetic, therapeutic and dignostic.

It is highly innovative to seek to know the genomic regions of canities by analysis of genetic link while other studies aim rather to decipher the bioctiimy of canities. The inventors have chosen to take advantage of the long-proposed hypothesis of the hereditary nature of premature canities (PC), or the appearance of white hair early in life. The family character of early hair whitening in some is indeed easily observable.

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. In order to guarantee the best chances of success for this identification of genes, 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. In addition, it is likely that, on the one hand, premature canities have a multigenic and not a monogenic origin, and that, on the other hand, environmental factors have an influence on the phenotype. It is a question of defining a set of causes predisposing to premature canities rather than a single mutation responsible for the phenotype. In this context, reverse genetics is not usually a technique recommended by geneticists. It is therefore original on the part of the inventors to have used this method.

The results of this work enabled the inventors to define chromosic and / or genomic zones comprising genes implicated with high probability in canities. In the present application, the regions of the chromosomes or the sub-parts of these regions, identified by the inventors as comprising genes statistically involved in canities, will be denominated indifferently "chromosomal regions of the invention" or "genomic regions of l 'invention "or" chromosomal areas of the invention "or" genomic areas of the invention ". As for the genes identified within the framework of the present invention within said regions, they will be called “genes of the invention”.

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.

For the fields of therapy and cosmetics, 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. GLOSSARY

In the context of the present invention, the terms stated have the following meaning:

By polynucleotide fragment is meant 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.

Although the base pairing is preferably of the Watson-Crick type, other types are not excluded, such as a Hoogsteen or reverse Hdogsteen type pairing.

We consider that the 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

3. by complementarity, or

4. by complementarity but by changing all or part of the Thymines to Uracil.

In addition, it is considered that 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 %.

This definition does not suppose that the two molecules are of the same nature, in particular as regards their skeleton, it is a correspondence between their sequence only.

For example, two identical DNA sequences "match" each other. Likewise, if these two sequences are substantially identical, that is to say identical more than 90%, they correspond to each other. An RNA sequence, resulting from the translation of any DNA molecule, "corresponds" to the sequence of this DNA molecule. Likewise, 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.

On the same diagram, we consider that the 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. 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

"Corresponding" if less than one error in 10 is introduced overall in the complementarity or identity processes, preferably less than one error in 100.

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. In the context of the invention, 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. The function of a DNA fragment also has other meanings in the present invention. In particular, 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.

Other functions can be envisaged for the DNA fragments. In particular, their mere presence within a gene can facilitate recombination. Similarly, a function according to the invention can be that of telomeres and relate to degeneration. Other specific functions attributed to DNA fragments are well known to biologists. A genetic marker is a detectable DNA sequence. In human genetics, 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. Among the conventional markers, there are two main categories which are microsatellite markers and SNP (Single nucleotide polymorphism).

A microsatellite is a repeated DNA sequence, made up of a relatively simple motif: a di, a tri or a tetra nucleotide most often. The number of repetitions changes for the same reason according to the individuals and can vary from a few units (a dozen at least for a dinucleotide), up to more than a hundred. These sequences are scattered almost everywhere in the genome, almost randomly, but in identical places from one individual to another. They are very abundant (about one every 10,000 nucleotides = 10 kb) and they are very polymorphic. It is the variation in length of the tandem repetition which constitutes the marker. These microsateUite sequences are therefore widely used as genetic markers.

Normally, there is no explicit link between a microsatelhte marker and a gene, except a co-location. The length of a tandem repetition does not, according to current knowledge, and apart from a few rare cases of intragenic markers associated with certain pathologies, have any link with the role of a gene. In the context of the present invention, 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.

There are different methods for defining the location of particular DNA sequences along the chromosomes. The physical unit of measure is the number of base pairs. However, 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. In particular, 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. As such microsatellite markers can be found approximately every 10 kb, it is therefore possible to define the position of any sequence to within 10 kb, by indicating the microsatellite markers surrounding it. A SNP (Single Nucleotide Pholymorphism) is a polymorphism that affects a single base of DNA. It is the most widespread form of polymorphism in the human genome, and it is also characterized by; high stability during transmission. Most of these polymorphisms have no functional implications. There are approximately 1 SNP on 100 base pairs. Knowledge of these SNPs makes it possible to establish a mapping of the human genome and the SNPs then serve as true markers of the genome, especially since they mutate slowly and have little chance of reappearing repeatedly.

By chromosomal region between two markers is meant the entire sequence between these two markers, the limits being understood, therefore the sequence of markers including.

In reverse genetics, 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.

The co-transmission of a phenotype and a marker suggests that the gene responsible for the phenotype and the marker are physically close to each other on the chromosome. 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. On the other hand, this study is based on statistics, these statistics being based on the analysis of a sample of the population, it is therefore a survey. Finally, it should be noted that when it is possible to associate a particular allele of the marker with an allele of the gene (in fact a phenotype), this association is a priori only valid for inter-family samples.

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.

By the term 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.

According to a first aspect, 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. According to a second aspect, 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.

According to a third aspect, 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 KIAA1042, CCK, CACNA1D, ARHGEF3, ALI 33097, KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETF1, HSPA9B, PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOF genes , G3BP, GLRA1, C5orf3, MFAP3, GALNTIO, FLJ11715, GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4 TBC105, LOC10 genes of the invention. For the three chromosomal zones identified above, 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. According to a possibility envisaged by the present invention, a fragment involved in canities or early canities and having a sequence meeting the requirements mentioned above can be used in therapy. More particularly, the first aspect of the invention relates to the genes of human chromosome 3 identified by the inventors as being involved in premature canities. According to this aspect, 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. According to this aspect, 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. According to this aspect, 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. These genes are included in the third chromosomal zone of the invention which is delimited on chromosome 11 by the microsatellite markers Dl 1S898 and Dl 1S925. 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.

According to preferred variants of the invention, reference is made to 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, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and FLJ11715, or all or part of a chromosome 3 gene chosen from the KIAA1042, CCK genes , CACNA1D, ARHGEF3 and AL133097: According to a preferred case, the fragment corresponds more particularly to any or part of an exon of one of these genes.

In what follows, will be designated "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.

For the genes identified by the inventors, 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.

For all the uses according to the invention in the field of cosmetics, the product of the invention can be packaged in different suitable forms, alone or in combination with other agents. In particular, 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.

Among the various cosmetic aims in the context of the present invention, 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. In particular, 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.

For the genes identified as “genes of the invention”, the present invention then relates to therapeutic uses in the field of pigmentation.

Conditions affecting the pigmentation system, whether that of the skin or that of the appendages, can have serious consequences on the health of those affected. Indeed, the pigmentation of the skin plays the role of protective barrier against light attacks in particular, people suffering from albinism are devoid of protection against the rays of the sun which constitute for them a significant danger. Other conditions involving pigmentation are also concerned with the present invention.

In the context of therapeutic and cosmetic uses which tend to modify a characteristic of the pigmentation, it is preferably pigmentation of the skin. According to other cases envisaged by the present invention, the type of pigmentation which must be modified relates to the pigmentation of the integuments, in particular the nails or the hairs.

According to a particular preferred case of the present invention, 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.

For. all the therapeutic uses, 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. Likewise, 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.

Likewise, 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. In particular, if 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. In both cases, 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. For introduction into cells, use may provide that 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. For therapeutic uses, the polynucleotide fragment is used in the manufacture of a medicament.

Concerning the chemical nature of this polynucleotide fragment, it 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.

Regarding its environment, 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.

As regards the regions framing this fragment, it is preferably under the control of regulatory sequences. If the fragment is inserted into a vector, 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. According to different cases envisaged by the present invention, 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.

According to other constructions envisaged by the present invention, the first use makes use of a polynucleotide fragment associated with a probe. This 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.

The 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

18 nucleotides constituting a sequence which corresponds to all or part of one of the 5 genes of the invention of human chromosome 3, or to all or part of one of the genes of invention of chromosome 5 or all or part of the one of the 18 genes of the invention of chromosome 11. In particular, a fragment according to the invention may contain only 18 bases complementary to 18 successive bases of one of the genes of the invention.

According to another particular case, 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. In the context of this first use, the number of polynucleotide fragments as defined above is not limited and is not necessarily limited to one.

In particular, use may be made of several polynucleotide fragments whose sequence corresponds, at least in part, to one of the genes of the invention within the chromosomal regions of the invention. Preferably, the 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.

This use can also allow the manufacture of a medicament for a therapeutic action, in the field of pigmentation. According to a particular case envisaged, the first use described involves a genetic modification, whether it is induced by a nucleotide fragment as described or not.

In the case where a gene responsible for pigmentation is defective because mutated, 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.

In the case where the activation of a gene is responsible for depigmentation, 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, in the field of therapy and in that of cosmetics, 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. For therapeutic uses, the agent thus defined is used in the manufacture of a medicament. Preferably, 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. Preferably, 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. Alternatively, 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. In particular, it may be a plasmid having only a short sequence corresponding to one of the genes mentioned. Preferably, the sequence correspondence is established on at least 18 successive nucleotides.

All of the foregoing remarks, in the definition part, on what is to be understood by “function of a DNA fragment” are valid for considering all the uses corresponding to a second use according to the invention. Given the plurality of functions of DNA fragments, the modulation of these functions includes very different aspects. In the particular case where the function of said fragment is to be transcribed, 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.

When the DNA fragment belongs to a regulatory sequence, 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.

In the context of this use according to the invention, as in the context of all the other uses according to the invention, 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.

However, in the context of this second use, 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. According to the first aspect of the invention, this region is that of the human chromosome 3 identified by the inventors. According to the second and third aspects of the present invention, 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.

Other agents according to the invention are preferably polypeptides capable of interacting with defined sub-regions of one of the genes of the invention. Preferably, 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. ",

According to a particular case envisaged, 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.

In the case where the activation of a gene is responsible for depigmentation, 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.

In the case where the inactivation of a gene is responsible for depigmentation, 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, in the field of therapy and in that of cosmetics, 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. For therapeutic uses, the agent thus defined is used in the manufacture of a medicament. Preferably, the DNA fragment has at least 18 nucleotides.

In particular, such 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. According to another case, an agent according to the invention modulates the function of a polypeptide resulting from the translation of one of the transcripts mentioned. Alternatively, 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. Preferably, 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.

Other preferred agents according to the invention belong to the class of polypeptides. In particular, the invention relates to proteins capable of binding to said transcript and thus of modulating its translation. Such agents from the class of polypeptides can be of natural or synthetic origin (synthesized chemically or by biotechnology). In particular, they may be antibodies. As mentioned above, this modulation can result in the action of promoting, preventing, delaying, accelerating or introducing errors in the translation of said transcript. In particular, 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. *

In the context of this third use, the number of agents modifying the function of an expression product as defined above is not limited and is not necessarily limited to one. - However, in the context of this third use, 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. According to the first aspect of the invention, this region is that of the human chromosome 3 identified by the inventors. According to the second and third aspects of the present invention, 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.

According to a particular case envisaged, 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. In the case where the activation of a gene is responsible for depigmentation, 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.

For therapeutic uses, the agent thus defined is used in the manufacture of a medicament. Preferably, the DNA fragment has at least 18 nucleotides.

In particular, 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. In the event of splicing, the transcript may therefore be smaller than the DNA fragment from which it originates. Preferably, if the expression product is an RNA molecule, it contains at least 18 nucleotides. According to another preferred case, 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. Preferably, a peptide expression product contains at least 6 amino acids.

The expression product according to the invention does not necessarily come from the steps of transcription or translation of genomic DNA. In particular, 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.

The use of an agent which is perfectly synthetic but similar to the expression product of an exogenous or endogenous DNA fragment corresponding at least in part to one of the genes of the invention is also envisaged by the present 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. In particular, to form 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. In particular, the invention relates to proteins capable of introducing a change in the functioning of the cell in which they act.

In the context of this use according to the invention, 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.

However, in the context of this fourth use, 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. According to the first aspect of the invention, this region is that of the human chromosome 3 identified by the inventors. According to the second and third aspects of the present invention, 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.

According to a particular case envisaged, the fourth use described involves a genetic modification, whether it is induced by an expression product of a DNA fragment as described or not. In the case where the activation of a gene is responsible for depigmentation, 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.

In the case where the inactivation of a gene is responsible for depigmentation, 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. For the four types of use described above in the context of the invention, the cosmetic uses are preferably in the field of pigmentation.

For the four types of use described above, 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.

The composition can be administered orally or applied to the skin (on any cutaneous zone of the body) and / or the scalp or the hair.

Orally, 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.

Depending on the mode of administration, 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.

For a topical application, 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. It 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. They can be anhydrous or aqueous. It can also consist of solid preparations constituting soaps or cleaning bars.

These 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.

When the invention consists in use for cosmetic purposes, 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.

When the composition is an emulsion, 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.

When the composition is an oily solution or gel, the fatty phase can represent more than

In a variant, 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. By way of example, 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).

In particular, a composition such as the coating of the product or products of the invention will have a diameter less than or equal to 10 μm.

In known manner, 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. 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.

As oils or waxes which can be used, mention may be made of mineral oils (petroleum jelly oil), vegetable oils (liquid fraction of shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils (Purcellin oil) , silicone oils or waxes (cyclomethicone) and fluorinated oils (perfluoropolyethers), beeswax, carnauba or paraffin. Fatty alcohols and fatty acids (stearic acid) can be added to these oils. As emulsifiers which can be used, there may be mentioned, for example, glycerol stearate, polysorbate 60 and the mixture of PEG-6 / PEG-32 / Glycol Stearate sold under the name Tefose® 63 by the company Gattefosse. As solvents which can be used, mention may be made of lower alcohols, in particular ethanol and isopropanol, propylene glycol.

As 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. Among these 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 plants such as those of Iridaceae or soy, extracts which may or may not contain isoflavones;

- 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;

agents for inhibiting hpoxygenase or inducing cyclo-oxidase promoting hair regrowth, such as those described by the Applicant in European patent application EP 0648 488;

- antibacterial agents such as macrolides, pyranosides and tetracyclines, and in particular lΕrythromycin;

- 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;

- steroidal or non-steroidal inhibitors of 5-reductases such as those described by the Applicant in European patent applications EP 0964 852 and EP 1 068 858 or else finasteride;

- ATP-dependent potassium channel agonists such as cromakalim and nicorandil.

In another possible implementation, the present invention relates to methods for diagnosing a predisposition to premature canities in an individual. Indeed, 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.

As it is very probable that environmental factors play a role in the phenotype "canities" as in that "early canities", it is a question, thanks to the methods of the invention, of evaluating the risks of developing such a phenotype, that is to say, a predisposition to premature canities.

A method according to the invention 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. By 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. Preferably, the marker is a SNP ('single nucleotide polymorphism').

According to a first aspect of a method of the invention, the marker or markers selected belong to the region of human chromosome 3 comprising the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097. Preferably, the markers are chosen as belonging to the sequence of one of these genes. According to a second aspect of a method of the invention, 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. Preferably, the markers are chosen as belonging to the sequence of one of these genes.

According to a third aspect of a method of the invention, 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. Preferably, 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.

Various markers are potentially preferred in the context of the implementation of the method of the invention. In particular, 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. Among the arαarqueurs 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. In many situations, 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.

When 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. According to the first aspect of a method of the invention, 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.

According to the second aspect of a method of the invention, 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.

According to the third aspect of a method of the invention, 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.

In particular, 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. In this case, 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. On the other hand, within the same family, some members of which were prematurely affected by canities, it is very likely that the cause of the susceptibility is unique. Therefore, during the comparison step, mentioned as a possible third step of the methods of the invention, 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. In particular, the invention relates to a method of identifying molecules capable of modulating the function of a polynucleotide fragment. According to the first aspect of the invention, 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. According to the second aspect of the invention, 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. Finally, according to the third aspect of the invention, 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.,

The various functions which a polynucleotide fragment can assume have already been explained in the present application. In particular, these functions depend on the nature of the polynucleotide fragment, depending on whether it is DNA or RNA for example.

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. In particular, 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. According to the first aspect 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. According to the second aspect of the invention, 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. According to the third aspect of the invention, 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. Preferably, 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.

As mentioned previously in the application, 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. For an RNA molecule, different stages of maturation are represented by the presence or absence of the cap, of polyadenylated tail, for example. By different 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. As for 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. As to what must be understood by the phrase "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.

Depending on the properties of said expression product used, those skilled in the art are able to determine which is the parameter, the variation of which is easy to follow. 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.

But as already mentioned, the inventors suspect that many genes are involved in the phenomena of pigmentation, and in those related to the regulation and cessation of this pigmentation.

Therefore, the particularly preferred uses are those which take advantage of the combination of the results obtained for the three chromosomal zones of the invention.

In particular, 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.

In addition, in another patent application, filed the same day by the same applicant, the inventors have also demonstrated, by a similar method, other genes also involved in the phenomenon of pigmentation or depigmentation, on chromosomes 6 ( between markers D6S1629 and D6S257) and 9 (includes marker D9S290 and the telomeric region) ,. This genetic basis enabled them to envisage uses in therapy and in cosmetics similar to those described previously, as well as diagnostic methods similar to those illustrated previously. Preferably, 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. 506, NT_007592.507, NT_007592.508, HSPA1B, G8, NEUl, NG22, BAT8, HLA-DMB, HLA-DMA, BRD2, HLA-DQA1, HLA-DQA2, NT_007592.588, GRM4, RNF23, FLJ22638, NT_007592. 459 and NT_007592.457, or that of a gene from human chromosome 9 chosen from the genes FREQ, NT_030046.18, NT_030046.17, GTF3C5, CEL, CELL, FS, ABO, BARLH1, DDX31, GTF3C4 and Q96MA6. Preferred genes are DDX31 and GTF3C4.

Preferably, among the various polynucleotide fragments which are used, 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.

All the preferred uses, the chemical nature of the fragments, their environment, have already been explained in detail in the section describing the first use according to the invention.

When at least two polynucleotide fragments are used, 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.

For therapeutic uses, 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_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. Preferably, among the various agents which are used, 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.

For therapeutic uses, the agents are used in the manufacture of a medicament. A third combination use 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 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, NT _. 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.

Preferably, among the various agents which are used, at least two modulate the function of the expression product of DNA fragments corresponding to two distinct chromosomes, or two distinct genes. It is also conceivable that for the same chromosomal zone of the invention, 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.

For therapeutic uses, the agents are used in the manufacture of a medicament. A fourth combination use 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 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, 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.

Preferably, among the various expression products which are used, at least two 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.

All the preferred uses of such expression products have already been explained in detail in the section describing the fourth use according to the invention. For therapeutic uses, the expression products are used in the manufacture of a medicament.

For the four types of combination uses described above in the context of the invention, the cosmetic uses are preferably in the field of pigmentation. Among the many combinations envisaged by the present invention, 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. . ; _< ^• ,

In order to take advantage of the combination of these chromosomal regions, the present invention also relates to combination methods. These methods are used to determine a possible predisposition to premature canities.

A combination method according to the invention 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, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650. Preferred markers belong to one of these genes. According to a variant of the method, 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). On chromosomes 6 and 9, 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. Preferably, among the markers selected, at least two do not belong to the same chromosomal region of the invention, to the same gene of the invention.

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 procedures for implementing these procedures have already been explained in the section devoted to diagnostic procedures.

For the four types of combination use described above, the combinations of the invention can be incorporated into a cosmetic or pharmaceutical composition as described above.

The combination methods of the invention are not limited to the use of two markers, nor are they limited to the two stages described and may contain other stages before or after the two stages mentioned.

In particular, 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. In this case, 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.

As with the diagnostic methods already mentioned in the present application, 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. Finally, 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.

According to a variant of the kit, the latter 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. 18, NT_030046.17, GTF3C5, CEL, CELL, FS, ABO, BARLHl, DDX31, GTF3C4 and Q96MA6. Preferred genes are the DDX31 and GTF3C4 genes.

Legend of figures

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.

Abscissa = position on the genetic map (0 = pter) Ordinate = NPL score

Figure 3A: Chromosomes 3 and 5; Figure 3B: Chromosomes 6 and 9

Figure 3C: Chromosomel 1 Figure 4: Schematic representation of the CP loci identified on chromosomes 11,

5 and 3 by the global genome study. The distance between markers is indicated in cM.

Figure 4A: Chromosome 11, locus 11 ql4-q22

Figure 4B: Chromosome 5, locus 5q31-q32 - ”.

Figure 4C: Chromosome 3, locus 3pl4.1-pl2.3

Figure 4D: Chromosome 6, locus6p21 -p 12 Figure 4E: Chromosome 9, locus 9q34

Figure 5: Lod scores simulated for the 29 families selected. _*

In order, 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.

The results are expressed by family. Figure 9: 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 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.

EXAMPLES

EXAMPLE 1

Summary of work done

In order to locate the gene (s) of premature canities (PC), a program of analysis of segregation (genetic bond) has been carried out in families where this trait is transmitted through the generations. After a series of preselections based on the statistical power of the sample and confirmation of the phenotypes, twelve families are selected to participate in a linkage study and the DNA was prepared from a sample of peripheral blood of each of the infoπnative individuals (presenting and not presenting the trait). The study was carried out according to two main approaches, analysis targeted on a candidate region and genome-global study on the twenty-two autosomal chromosomes and the X chromosome.

From all the analyzes carried out by fixing or not fixing parameters for the transmission of the CP trait, 3 potential loci emerge on chromosomes 11, 5 and 3 in addition to those identified on chromosomes 6 and 9. The three loci (chromosomes Ilql4-q22, 5q31-q32, 3ρl4.1-pl2.3) show suggestive signs of CP binding.

This study, and in particular the discrepancy between the scores obtained for the parametric / non-parametric analyzes, suggests that premature canities would not be caused by a small number of major effect genes, but would rather be governed by a multifactorial system including the action of several predisposition genes. INTRODUCTION

The hereditary nature of premature canities (PC), or the appearance of white hair early in life, has been a hypothesis proposed for a long time, due to the family nature of early whitening of hair in some people.

To explore canities due to their genetic aspect, a DNA segregation study was carried out in families whose canities appear early in life. In order to guarantee the best chances of success for this gene hunt, the composition of the sample for the study was carried out according to a rigorous protocol for the attribution of the phenotype and the selection of families. The CP phenotype was attributed to the only individuals who presented gray hair before 25 years and whose half of the hair was gray at 30 years. The families were selected for the study on. the base; of their statistical performance in the segregation analysis.

The part of the study rehearsed is described according to four main periods:

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.

The results obtained at each epoch are presented in the form of tables and figures in a synthetic way in the summary tables or in a deep way in the detailed tables. RESULTS

A- Epoch 1: Choice of families with the help of link analysis simulation

Following an attempt to select families with premature canities according to informativeness criteria for gene localization, 29 families were subjected to a genetic hedgehog analysis analysis. Based on the availability of all individuals, it turned out that this project had very encouraging success potential because nineteen pedigrees (or 255 individuals) were then selected. This conclusion is only valid if the phenotypes are confirmed and if the majority of subjects agree to participate in the study. Among this selection are seven very informative families who individually could reach or exceed a Lod score Z≈4.00 (more than the lower limit of significance of the Lod score which is Z = 3.0Q). In order to gather the maximum chance of success, it was very important that the diagnosis of PC be assigned with rigor.

The result of this study allows, depending on the robustness of the clinical evaluation, to qualify the families which are then collected for the genetic study.

1. Criteria for assigning the CP phenotype

Twenty-nine families among the 65 were selected, based on structural criteria (total number of individuals, affected, available), to be analyzed in simulation. During the simulation process: 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. 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. In addition to the codes indicating for each individual, the family ties, the sex, the phenotype as well as the genotype which will be incremented by the SLENK simulation software, 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 phenotype of each individual was assigned according to the information in the pedigrees and description tables of the Genormax report. However, for some individuals, the phenotype was modified according to the criteria indicated in table 2. The individuals, not present on the initial pedigrees (identified by a number only) were carried phenotypically unknown, and are considered unavailable (cd = 3).

at. Availability codes

During the simulation, only the individuals for whom (Table 1) were taken into account:

- it will be possible, according to the Genormax study, to draw blood in order to prepare DNA for genetic studies (age, domicile in metropolitan France / overseas / foreign, prior consent);

- the phenotype of premature canities will have been clearly defined (Table 2).

Table 1: Definition of availability codes

Table 2: Definition of phenotypes

b. Assigning the phenotype by age

In order to avoid the risk of errors, 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.

vs. Other parameters

After examining the variation of the maximum Lod score in the Can65 family (test 100, 200, 300, 500 replications), the number of replications (generations of allelic combinations) 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.

2. Results

a- Classification of families according to their scores

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.

In genetic link analysis, to be significant, the Lod score must reach or exceed the value of 3 (result at 1000/1). b- Effect of an incorrect diagnosis on the results of link analysis

The effect of assigning an incorrect diagnosis was tested on the analysis results (in the event of binding to a locus) by a simulation on the Can46 family by varying the phenotype of 1,2 or 3 individuals (table 4).

Table 4: Lod score obtained for a series of distances from the marker to the trait locus (Lod score maximum in bold).

1- according to the current diagnosis (maximum lod score) distance 0.0 0.01 0.05 0.1 0.2 0.3 0.4 Lod score \ 2.28 2.24 2.08 1.88 1-44 0.95 0.43 a

2- 3 incorrectly diagnosed individuals (A2, RIO, R19) distance 0.0 0.01 0.05 0.1 0.2 0.3 0.4 Lod score -3.89 -1.75 -0.90 -0.50 -0.14 -0.01 0.01 a 3- 2 incorrectly diagnosed individuals (A2, R19) distance 0.0 0.01 0.05 0.1 0.2 0.3 0.4

Lod score -2. 85 -0. 75 -0. 05 0 .22 0.36 0 .30 0. 17 a

4- 1 incorrectly diagnosed individual (R19) distance 0. 0 0. 01 0. 05 0. 1 0. 2 0 .3 0 .4

Lod score 1.24 1.24 1 .23 1. 16 0. 94 0. 63 0. 27 a

3. Discussion, conclusion and decisions

This simulation study allows the 29 pre-selected families to be placed in 5 groups according to the maximum potential Lod score. Although a link is significant as soon as the value of Lod score of Z = 3.00 is reached, the inventors preferred to distinguish 2 groups when this criterion is verified because the maximum simulated Lod score is very rarely reached. So the probability that the real Lod score for families in group B (3 <Zmax <4) is quite low.

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.

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.

It seems, subject to a robust clinical characterization, that the families of groups A, B and C are suitable for a genetic hedge analysis study and that they must be collected (individuals with cd = 2). In fact, genetic linkage analyzes performed on insufficiently characterized samples are doomed to failure or to a "pale" result (imprecise locus). Given that in such analyzes, certain parameters cannot be under total control (in particular the informativeness of the genotypes), and that the genetic heterogeneity always possible makes the task more delicate (because it reduces the power of the analysis on all families), it therefore seems essential to gather all the assets that can be managed from the start. In this first step, the inventors therefore strongly recommended that the phenotype of each individual with an appropriate availability code (cd = 2) be carefully checked before collection (confirmed phenotype, or exclusion from the sample / family).

B- Period 2: Collection of samples, confirmation of phenotypes and new estimates of the study's potential

On the basis of the results of epoch 1, the 19 families (groups A, B, and C) selected to form a bank from which the pedigrees for the genetic linkage analyzes were contacted for confirmation of PC diagnosis and collection of a series of affected and unaffected individuals.

This phenotype medical verification campaign helped to confirm many of the PC diagnoses, but not all as expected. The refusal of a few key individuals (with PC) to participate in the project, the death of some as well as the readjustment of part of the phenotypes led to not retaining a certain number of families and reduced the informativeness potential of several others families.

1- Re-estimation of the potential of the study after confirmation of the phenotypes

In order to re-estimate the potential of the study after verifying the phenotypes, the inventors simulated a hedge analysis on the 8 families; which could still be informative. 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 5: Potential Lod scores as a function of the rate of genetic heterogeneity in PC

The detailed results by families are shown in the table in Figure 6.

Rate'

Heterogeneity Medium Si; dDev Minimun Maximum Max epoch1

0% 5.094620 1. .679698 1.221207 8.835722 38.823

50% 1.619190 1. .553049 0.000000 7.409957 -

70% 0.835383 1. .022004 0.000000 5.517145 _

2- Conclusion

In a continuous effort to optimize the sample for the linkage analyzes, 4 additional families have been identified (Table 6; 2 families -can65B and can46B- are collateral branches of the families can65 and can46) and the new phenotypes checked. Table 6: Final list of families

1 C103974

2 F104512

3 CAN33

4 CAN35

5 CAN43

6 CAN46

7 CAN46B

8 Çan53 ,.

9 CAN55

10 CAN62

11 CAN65

12 CAN65B

After a new simulation (Table 7), it turns out that the general maximum Lod score for binding is barely higher (Z = 8.91) if the 12 families selected (strictly defined phenotypes) are linked to the same locus. The expected increase in the Lod score by the contribution of new families is however reduced by the correction and hardening of the phenotypes.

Table 7: New simulation after collection of new families. The potential Lod scores are expressed as a function of the rate of genetic heterogeneity.

The detailed results are shown in the table in Figure 7.

Rate'

Hey erogeneity Medium StdDev Minimun Maximum

0% 4.752321 1.748924 0.356854 8.914062

50% 1.535356 1.418022 0.000000 6.078132

70% 0.719737 0.978920 0.000000 5.282466

The power of the sample can also be observed in terms of the number of replications which reach or exceed the Lod scores Z = 1.0, Z = 2.0, Z = 3.0 respectively and which gives an approximation of the chance of finding a significant connection. (table 8). Table 8: Probabilities (in%) of reaching or exceeding a Lod score of 1, 2 or 3 for each rate of 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

C- Period 3: Analysis of genetic linkage of the PC with the candidate region

1- Hypothesis 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.

2- Final composition of families

For the sake of technical optimization, a sample of 92 affected and non-affected individuals among the 12 families is selected (see Figure 1). This selection is formed based on the potential informativeness of each individual and confirmed by a new link simulation. The addition of a few individuals leads to a slight increase in the potential Lod score (from 8.91 to 9.04, depending on complete homogeneity, Table 9) and therefore in the power of the sample (Table 10).

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.

Heterogeneous rate :. .té Medium StdDev Minimun Maximum

0% 4,367,608 1,649,267 0.564,761 9.042465

50% 1.354325 1.359845 0.000000 6.610168

70% 0.666549 0.891330 0.000000 4.988076

90% 0.222622 0.378039 0.000000 2.528617 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.

Heterogeneity rate 0% 50% 70% 90%

Lod score

1,000 98, .000 48,500 21,500 4,000

2,000 94, .500 23,500 9,500 0.500

3,000 79, .000 12,000 3,000 0,000

3- Microsatellite markers used •. The distribution of markers on the candidate region of chromosome 3 is presented in Figure 2.

4- Link analysis

Several types of linkage analyzes have been carried out in order to increase the probability of observing an existing hedge between this chromosomal region and the PC. For this analysis, the following two approaches were used:

- 2-point (iterative analysis between the line and the markers taken one by one);

- multipoint (analysis for each chromosome according to a map of markers placed according to their respective distances).

1. Analyzes with defined, parametric parameters (PL): Transmission mode (dominant), line frequency (1%), equifrequency of alleles of markers tested and penetrance (90% mutant heterozygotes - 100% mutant homozygotes). 2- point and multipoint method.

2. Independent analysis of the mode of transmission of the trait, non-parametric (NPL): Deviation analysis of the proportion of shared alleles for each pair of affected individuals (in each family by identity by descent) compared to transmission to hazard. In the multi-point analysis, all the pairs of impairments (ail-peers, Z-all≈logio score of p-values, and p-values) were considered. In the 2- point method, it is the pairs of germains that have been studied (affected sib-pair, p-values).

5- Results The results are set out in Table 11. Table 11: Result of the linkage analysis on the candidate region

When only a position is indicated, and not a marker name, it means that the position is intermediate between 2 markers.

6- Discussion and conclusion

This study distinguishes a strongly suggested locus of predisposition to premature canities on chromosome 3pl4-pl2 (towards markers D3S2409 and D3S1766; multipoint NPL = 2.58 at position 12 and HLod: = 1.55 at position 24).

This result positively documents the presumptions of association which relate to the chromosomal region 3pl4-P12 with respect to premature canities.

It should be noted that one family (CAN53) shows a relatively high hedge for this chromosomal region.

D- Epoch 4: Analysis of the global genetic link between PC and the genome.

The global analysis of the genome makes it possible to make a visit of all the chromosomes (a global survey) in order to find regions involved and possibly a major locus which would govern early canities. This large analysis also makes it possible to estimate the rate of genetic heterogeneity in PC.

1- Content of families

These are the same families as those that were studied in the candidate regions region (epoch 3), but with some adjustments in their content. (see table 12). Some uninformative members have been replaced by others, more recently recruited, or whose diagnosis was clarified later.

In order to confirm the benefit of the evolution of the sample, a new simulation of hedge analysis was performed for different situations of genetic heterogeneity (Table 13 and tables in Figures 11A, 11B, 11C and 11D). The Lods scores expressed show a favorable development. Thus, considering the possible average Lod score, it would be possible to obtain a significant result (Z> 3.0) for heterogeneity reaching 20% (i.e. 1/5 of families not linked to the locus). In fact, this result is very conservative and it would be possible to reach significance with a clearly more heterogeneous sample (ie 50-70% with the use of microsatellite markers showing an average heterozygosity of 0.7).

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)

families Candidate Region Genome Global

A35 11 11

A46 12 12

A65 5 9

A53 8 9

B43 7 7

B55 7 7

C33 6 6

C62 6 6

A46B 6 7

A65B 6 6

103 974 12 10

104512 6 6

Total individuals 92 96 Table 13: Potential Lod scores as a function of the rate of genetic heterogeneity in PC

The detailed results are shown in the tables in Figure 11.

Average heterogeneity rate StdDev Minimun Maximum

0% 4.751841 1.749689 0.334792 9.338889

20% 3.115806 1.866821 0.024841 8.780452

50% 1.378378 1.467071 0.000000 7.508479

70%, 0.672997 0.904472. 0.000000 5.226281

* 90% 0.242418 0.402384 0.000000 2.722478

The power of the sample can also be observed in terms of the number of replications (of, genotypes) which reach or exceed the respective Lod scores Z≈l.0, Z = 2.0, Z = 3.0. 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

The detailed results are shown in the table in Figure 12.

Heterogeneous rate: ity 0% 20% 50% 70% 90%

1,000 99,000 87,500 47,000 23,000 5,500

2,000 93,500 69,500 25,500 11,500 0.500

3,000 84,000 48,500 15,000 3,500 0,000

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. 2- DNA markers

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) „from the Généthon collection (Evry, France).

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.

3- Linkage analyzes

For this global approach, 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. For each of these analyzes, 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). a- Analyzes with defined, parametric parameters (PL) • Transmission mode (dominant),

• Line frequency (1%),

• Allelic equi-frequencies of all markers,.

• Defined penetrations (90% mutant heterozygotes - 100% mutant homozygotes), • 2-point and multipoint methods.

• Link probability scores are expressed in:

- Lod score Z (homogeneous sample); - ZH Lod score (heterogeneous sample) and heterogeneity rate α (proportion of families which are not linked to this locus).

b- Independent analysis of the mode of transmission of the trait, non-parametric (NPL),

It is an analysis of the deviation in the proportion of alleles shared by pairs of affected individuals compared to a random transmission of alleles (identity by descent). The inventors considered all the pairs of affected individuals for the multi-point analysis, and the pairs of siblings for the 2-point analysis. Binding probability scores are expressed in

- NPL or Z-all (Log10 of p value) for the multipoint method on all pairs of affected individuals; - "p" value for the 2-point method on the affected germain pairs. 4- Results

Figure 3 reports the NPL scores obtained for the analysis of non-parametric hedge on chromosomes 3, 5, 11, 6 and 9.

a- detailed results

Tables 16 (best 2-point analysis results) and 17 (best multipoint analysis results) report the best results obtained for the chromosomes selected.

Table 16: Best 2-point analysis results for the chromosomes selected

PL NPL

Chromo Localization cM

Locus * Marker Z (t) Theta Z (a, t) Theta Alpha Sibpair some (distance -pter)

Homogeneity Heterogeneity

3 4 D3S1285 91.2 0.688 0.2 0.819 0.12 0.59 15 D3S1601 214.4 0.006153

5 6 D5S422 164.2 2.007 0.14 2.007 0.14 1 23 D5S647 74.07 0.021524

11 19 D11S908 108.6 0.777 0.2 0.846 0.06 0.6 4 21.5 0.203028

Table 17: Best results of the multi-point analysis for the selected chromosomes

b- retained results

Table 18 reports the best results retained for each type of analysis (PL, NPL) discussed below:

Table 18: Best results retained for each type of analysis (PL, NPL)

2-point (2P) gold

Chromosome Position / pter Score Multi anointed (MP)

Non-parametric

2-poini (2P) gold

Chromosome Position / pter Multipoint Score (MP)

parametric

i In terms of PL: a- A Lod score (2P) ZH = 2.007 was noted on the long arm of chromosome 5 (position 164.2 cM from the upper telomer), ie in position 3/4 on the band 5q31-q32. b- Lod intermediate scores (1.5 <ZH <2.0)

- chromosome Ilql4-q22, position 106 (MP-ZH = 1.53) c- Lod score of interest (1.0 <ZH <1.5)

- chromosome 5q31-q32, position 168 (MP-ZH = 1.11)

- chromosome 6p21-pl2, position 61 (MP-ZH 1.43) - chromosome 6ql3-ql4, position 90 (2P-ZH = 1.46) .- -

ii In terms of NPL:

A distinction has been made here between log ₁₀ scores for the study of deviation from the Allechic sharing, identity by descent (IBD), for all the pairs of affected (Z-all multipoint study scores) as well as the p values for the pairs of first affected (affected sib-pairs, 2-point study scores, p-values).

The best scores are discussed according to their rank in relation to the significance limit: - Z-all> 3, 2.5 <Z-all <3, 2.0 <Z-all <2.5

- p <10 ^"5 and 10 ^{" 5} <p <10 ^"4

a- Z-all scores> 3.0

On chromosome 6p21-pl2, the score reached using the genome-global markers maximizes to a Z-all = 3.52 at position 71 (between markers D6S1610 and

D6S257). However, the accuracy of the locus is probably affected by the great distance between these 2 markers of the genome-global battery which is more clearly greater than the mean interval observed (26.11 cM).

The second best score is reached on chromosome 9q31-q32, position 151 (Z-all = 3.37) b- Scores 2.5 <Z-all <3.0

- chromosome 3pl4-pl3, position 72 (Z-all = 2.62)

- chromosome Ilq21-q22, position 106 (Z-alb = 2.61) c- Scores 2.0 <Z-all <2.5

- chromosome 9q31-q32, position 131 (Z-all = 2.13)

- chromosome 3q21, position 101 (Z-all = 2.15) as well as the p-values <10 ^"5 and 10 ^{" 4}

d. p <lxl0 ^'4

- chromosome 6q31.3-q33, position 154 (p = 0.000012) iii The loci which are identified simultaneously by PL and NPL:

5- Discussion and conclusion i.

The 2 significant scores or at the edge of significance depending on whether the trait is considered to be monogenic or multifactorial (Lander and Kruglyak 1995) were observed for chromosomes 6ρ21-pl2 (NPL MP Z-all = 3.59) and 9q31 -q32 (NPL MP Z-aU = 3.37). Among these 2 loci, the most robust is that of 6p21-pl2 which is reinforced by an MP-

PL Lod score, which although average, maximizes at ZH = 1.42.

Another locus also appears quite interesting, it is located on the chromosome

Ilql4-q22 because the PL and NPL scores maximize at the same position 106 (Z-all 2.61, PL

1.52). 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).

Finally, the locus 5q31-q32 with the best PL lod score (2P) (ZH = 2.00) also lies in suggestivity values (in monogenism).

A series of p-values must be added for analyzes of affected siblings who are also in the suggestivity range (chromosomes 6q31.3-q33). These are loci to consider too. D- General discussion and conclusion

At the end of the different periods of analysis, several chromosomal regions are identified or suggested. Validity of loci Uql4-q22, 5q31-q32, 3pl4.1-pl2.3

a Chromosome Ilql4-q22

The inventors identify a region between positions 100 and 115 (between Dl 1S898 and Dl 1S925) (Figure 4A).

b Chromosome 5q31-p32

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. By placing this distance from D5S422 towards the top of the map (position 149), we arrive in the vicinity of the locus which brings together the best multi-point NPL score on chromosome 5 (Z-all = 1.70, towards marker D5S436). A certain consensus appears for this locus also (Figure 4B).

c Chromosome 3pl4.1-yl2.3

It is a region of almost 30 cM between positions 60 and 87 (between D3S1277 and D3S1285 (Figure 4C).

EXAMPLE 2 Analysis of the regions of interest with SNPs 'Single Nucleotide Polymorphism'

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.

Analysis of the entire genome of families in which the premature canities trait segregated has indeed revealed 5 chromosomal regions which are related to the phenotype. Beyond loci 6p21-pl2 (A) and 9q31-q32 (B) distinguished by significant Lod scores (non-parametric Lod score NPL = 3.59 and NPL = 3.36 respectively) other regions are of interest in reason for suggestive link scores in more than one analysis.

The region on chromosome 3 (3pl4.1-pl2.3) indeed presents results of equivalent amplitude obtained simultaneously in the study of candidate genes and global genome (multipoint candidate regions Lod non-parametric score RC-MP- NPL = 2.58, candidate regions 2-point analysis Lod parametric score RC-2P-PL = 1.55, global multipoint genome Lod non-parametric score GG-MP-NPL = 2.62).

The region on chromosome 5 (5q31-q32) is chosen because of the only locus in PL-2 point analysis which reaches a significant Lod score (GG-2P-PL = 2.00). The region on chromosome 11 (1 Iql4-q22) is chosen because of its score among the 5 highest in NPL and in PL (GG-MP-NPL = 2.61, and GG-2P-PL 1.52).

Abbreviations used

RC: candidate regions

GG: global genome MP: multipoint

2P: 2-point analysis

NPL: Non-parametric Lod score

PL: Parametric lod score

SNPs (single nucleotide polymorphism) represent a form of polymorphism particularly widespread in the human genome and very stable. 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. .

At the end of the Human Genome Project program, SNPs are now better known and referenced, as well as their position in the genome (GDB). Different methods have been developed to demonstrate these polymoφhisms between different individuals, often based on the methods used to detect point mutations (RFLP-PCR, hybridization with allele specific oligomers, mini-sequencing, direct sequencing ...). ). In the context of the present application, the inventors have used MALDI-TOF technology (matrix-assisted laser desoφtion / ionization time-of-flight mass spectiometry) to detect the various alleles of the candidate SNPs. Further details on this technology are known to those skilled in the art and are described in various publications (Stoerker J, et al, Nat Biotechnol 2000 Nov; 18 (ll): 1213-6 and Tang K et al, Proc. Natl. Acad. Sci. USA 1999 Aug, 96, 10016-20).

First, the inventors defined very precisely the regions of chromosomes 3, 5 and 11 to be analyzed with SNPs. In a second step, 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. In a next 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.

1- Definition of regions to be analyzed by SNPs

Initially, 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).

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 (in terms of bp base pairs) 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).

UCSC Freeze (correspondence with NCBI Assembled Genome Sequence version, build)

November 2002 (NCBI Build31) June 2002 (NCBI Build30)

April 2002 (NCBI Build29) December 2001 (NCBI Build28)

2- Search for candidate SNPs (in silico) and validation (experimental). Besides regions C, D and E as defined above, a second step consisted in determining a collection of SNPs belonging to these regions, so as to obtain a map of markers of the three regions. These markers have also been defined in such a way that they cover the 77Mbp (total length of the three regions) in a homogeneous and equidistant manner. The distance between the various SNPs has been set at an average of 50 kb. This operation was carried out by the selection of 3848 SNPs meeting these criteria (SNPs candidates in silico). Among the 3848 thus envisaged during the first stage, 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).

3- DNA assembly (Pooling)

In order to increase the genotyping capacity, a pooling strategy was carried out on the different DNAs. The power of this method is reported in various publications (notably Werner et al, Hum Mutât 2002 Jul; 20 (l): 57-64, Bansal et al, Proc Natl Acad Sci USA 2002 Dec 24; 99 (26): 16871- 4). .

To carry out this pooling, 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.

Initially, two kinds of criteria were defined, the primary criteria to which are assigned score values of 2, and the secondary criteria to which are assigned score values of 1.

There are 2 primary criteria (score value = 2 for each of them) which are: (i) first gray hair before 18 years of age; (ii) hair with pepper and clear salt at 30 years.

There are 3 secondary criteria (score value≈l for each of them) which are: (i) first gray hair before 25 years of age; (ii) Dark pepper and salt hair at 30, (iii) family notion of premature canities.

By adding up for each individual the scores obtained with each of the diagnostic criteria, one can define for each individual an intensity score of the premature canities phenotype. It was thus possible to define several different groups according to the phenotypic score.

Among the affected individuals, 72 individuals whose score is greater than or equal to 4 or 5 and 132 individuals whose phenotypic score is greater than or equal to 2.

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: these 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.

In this way, in addition to the matching achieved versus not reached by the phenotypic trait (CP), 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 constitution of the different groups is shown diagrammatically in FIG. 13.

The use of these rigorous methods for clinical diagnosis of affected and contagious subjects gives a guarantee of reliability on the quality of the phenotypic data.

Furthermore, 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.

4- Selection of SNPs validated for genotyping on grouped DNAs.

Different groups of SNPs were formed according to the following definition: Definition of groups 1, 2, 3 and 4

- group 1: lower allele> 10%, standard deviation <0.025

- group 2: lower allele> 10%, standard deviation> 0.025 - group 3: lower allele <10%, standard deviation O.025

- group 4: lower allele <10%, standard deviation> 0.025

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. By this method, 3227 SNPs were thus validated.

Among the 3,227 validated SNPs, 1,264 were chosen during a new selection stage. This new selection is based on the following criteria:

- intragenic or close to gene regions

- average inter-SNP intta gene intervals between 30 and 50 Kb.

The vast majority of SNPs were chosen from group 1, which represents the greatest reliability. In order to better cover the regions to be analyzed, the overall size of which is 77 Mb, the analysis has been concentrated on the gene regions and regions close to the genes likely to contain regulatory sequences, leaving aside the regions without genes. This makes it possible to increase the density of the coverage (decrease the inter-SNP intervals) in the coding regions and therefore to increase the probability of having several positive SNPs in areas which would be associated with tiait CP (early canities). By gene regions is meant both the regions containing known genes, as well as that likely to contain them (predicted genes).

5- Allélotyping on assembled DNA. For the 1264 SNPs selected in step 4, 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.

However, since the experiment is carried out on "pools" and not on an individual DNA, only the 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.

The experiments were repeated 3 times (3 PCR), each of the three PCRs being then tested 5 times on MALDI-TOF in order to obtain a reliable average value. Only the results for the groups of 72 CPs and their controls were considered, these seem indeed more homogeneous (the comparisons of the AU-BII groups providing only a few positive results, raising fears of the presence of false negatives). The frequency of the two alleles for each of the SNPs are calculated in the different grouped DNAs. However, genotypes are not determined in this type by this study and therefore their frequencies among the different groups will not be available. The statistical significance of the allelic frequency differences between the groups is estimated by a "p" value. Only SNPs whose pvalues are less than 0.05 (5%) were selected.

a- Table of allelotypic comparisons between groups.

The following tables illustrate in tabular form the results obtained.

Definition of columns:

-CHROM: chromosome number

-CHROMPOS: position on the chromosome (NCBI build 30)

-SNP m: Name of SNP used

-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.

-LD / freq: 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.

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.

This linkage disequilibrium analysis made it possible to define DNA blocks which are materialized by several markers whose co-segregation of the alleles deviates from a co-segregation governed by chance alone. This situation is produced by an absence or a deficit of recombination within this block. The size of the regions exhibiting a linkage imbalance varies according to the chromosomal regions, it seems to extend over 10 to 200 kb.

When the area is black in this column, the association is significant. In gray, the association values are marginally good (acceptable). - p-val-eval: This criterion is based on the p-value alone. If the p-value of an SNP or of several grouped in clusters is less than 1%, then the cluster is marked as important: black box.

Only SNPs with a p-value of less than 5% have been indicated in the table, with the exception of negative SNPs located between two positive SNPs, which determines a double spot and those framing a cluster of interest.

REGION C

?

REGION E

The table below summarizes the number of positive SNPs within each region, depending on whether they are: -Spots:

- Isolated positive SNPs - Positive double SNPs (example: 2 positive SNPs separated by 1 negative SNP) - Clusters:

-2 contiguous positive SNPs

- More than 3 contiguous positive SNPs - Link / frequency imbalance

* LD: Linkage desequilibrium Results

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

(link imbalance) and combined allelic frequencies reveals only 3 areas of interest. Of these 3 zones 2 loci (positions 41527287-41677819 and 55638663-56042041) are characterized by a p-value <1%. The gene content of these 3 areas (Cl, C2, C3) is developed below:

Cl (41527287-41677819V.

- Hypothetical protein KIAA1042

-CCK: gasτrin / cholecystokinintypeb receptor (cck-b receptor) C2: f52846896-5291394n: -CACNA1D: voltage-dependent 1-type calcium channel alpha-ld subunit

C3: (55638663-56042041):

-ARHGEF3 rho guanine nucleotide exchange factor 3; rhogef protein; 59.8 kda protein; exchange factor found in platelets and leukemic and neuronal tissues, xpln. - Hypothetical protein ALI 33097

ii ~ region D 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.

The gene content of these 7 zones is developed below: Dl (136479801-137007868):

-KLHL3: kelch-like protein 3

-HNRPAO: heterogeneous nuclear ribonucleoprotein aO (hnrnp aO).

D2 (137542040-137771805)

-CDC25C: map / microtubule affinity-regulating kinase 3 (ec 2.7.1.27) -EGR1: early gro th response protein 1 (egr-1) (krox-24 protein)

-C5orf6: predicted

-C5orf7: predicted

-LOC51308: predicted

-ETFl: eukaryotic peptide chain release factor subunit 1 (erfl) -HSPA9B: stress-70 protein, mitochondrial precursor

D3 (139931847-140118601)

-PCDHAI to PCDHAI 3: protocadherin alpha 1 precursor to protocadherin alpha 1 precursor

D4 (149518721-149586774) -CSF 1R: Macrophage Colony Stimulating Factor I Receptor Precursor

-RPL7: 60s ribosomal protein 17

-PDGFRB: beta platelet-derived growth factor receptor precursor (ec 2.7.1.112)

D5 (149793126-149995886)

-TCOFl: Treacle Protein (Treacher Collins Syndrome Protein). -ALI 33039: predicted

-CD74: hla class ii histocompatibility antigen, gamma chain

-RPS14: 40s ribosomal protein s 14

-NDSTl: Heparan Sulfate N-Deacetylase N-Sulfotransferase (Ec 2.8.2.8) D6 (151235618-151373121)

-G3BP: ras-gtpase-activating protein binding protein 2 -GLRAl: glycine receptor alpha-1 chain precursor D7 (153463449-153854650) -C5orf3: predicted

-MFAP3: microfibril-associated glycoprotein 3 precursor

-GALNTIO: putative udp-galnac: n-acetylgalactosaminyltransferase polypeptide

-FLJ11715: predicted

iii- region E ^• • -

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,

E5, E6) are more promising due to the significance of the comparison between samples. The gene content of these 6 areas is developed below:

El (108893187-108944206)

-GUCY1A2: soluble guanylate cyclase, alpha-2 chain (ec 4.6.1.2) E2 (110056711-110546142)

-CUL5: 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;

-ATM: serine-protein kinase atm (ec 2.7.1.37) (ataxia telangiectasia mutated

-AF035326: predicted -AF035327: predicted

-AF035328: predicted

-BC029536: predicted E3 (115527211-115745012)

-FLJ20535 -DRD2: d (2) dopamine receptor

-ENS303941: predicted E4 (117397672-117752160):

IGSF4: immunoglobulin superfamily, member 4; nectin-like protein 2 E5 (118532530-118685957)

No known gene

E6 (119417270-119469358)

-LOC51092: predicted -BC010946: predicted

-TAGLN: transgelin (smooth muscle protein 22-alρha) (sm22-alρha) (ws3-10) (22 kda actin-binding protein).

-PCSK7: proprotein convertase subtilisin kexin type 7 precursor (ec 3.4.21.-) -ENS300650: predicted

EXAMPLE 3

Example of Compositions

- hair lotion DNA fragment from the chromosomal area between the markers D3S1277 and D3S1285 0.5 g

Propylene glycol 20 g

Ethanol 95 ° 30 g

Water qs 100 g

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.

There is then a decrease in the appearance of white or gray hair and a repigmentation of gray hair.

- shampoo treating DNA fragment from the chromosomal area between the markers D5S2115 and D5S422 1.5 g

Polyglyceryl 3-hydroxylarylether 26 g Hydroxy propyl cellulose sold under the name Klucell G by the company Hercules 2 g

Preservative qps

Ethanol 95 ° 50 g

Water qs 100 g 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.

- Gel treating DNA fragment from the chromosomal area between the markers D11S898 and D11S925 0.75 g

Eucalyptus essential oils 1 g

Econozole 0.2 g

Lauryl polyglyceryl 6 cetearyl glycoether 1.9 g

Preservatives qs Carbopol 934P sold by BF Goodrich Corporation 0.3 g

Neutralizing agent qs pH 7

Water qs 100 g

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.

Bibliographic references

• E. Lander and L. Kruglyak. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results [see comments]. Nat.Genet. 11 (3): 241-247, 1995.

Claims

claims

1. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D,

5 ARHGEF3 and AL133097, for cosmetic purposes, in the field of pigmentation, said fragment having a length of between 30 and 5000 nucleotides.

2. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene

10 of human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D,

ARHGEF3 and ALI 33097, for the manufacture of a medicament for therapeutic use in the field of pigmentation, said fragment having a

^{* "'} " length between 30 and 5000 nucleotides. ^*

3. Use according to claim 1 or 2, characterized in that said fragment has a length of between 50 and 3000 nucleotides.

4. Use according to claim 1 or 2, characterized in that said pigmentation is that of the hair.

5. Use according to claim 1 or 2, characterized in that it relates to the prevention or treatment of canities.

6. Use according to claim 5, characterized in that said canities is an early canities.

7. Use of a polynucleotide fragment according to claim 1 or 2, characterized in that said fragment is associated with a fluorescent, radioactive or enzymatic probe. 25

8. Method for the diagnosis of a predisposition to premature canities in an individual, comprising the following steps: i) selection of a marker belonging to a gene of human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097, 30 ii) determination of the alleles of the chosen marker present in a sample of genetic material of said individual.

9. The method of claim 8 comprising the additional step iii) comparison of the algebraic form of the marker with that of other individuals to establish the diagnosis.

10. Method according to claim 9 characterized in that the other individuals are members of the same family as that of the individual to be diagnosed.

11. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from 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,

GLRA1, C5orf3, MFAP3, GALNTIO and FLJ11715, for cosmetic purposes, in the field of pigmentation, said fragment having a length of between 30 and 5000 nucleotides.

12. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from 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 and FLJ11715, for the manufacture of a medicament for therapeutic use in the field pigmentation, said fragment having a length of between 30 and 5000 nucleotides.

13. Use according to claim 11 or 12, characterized in that said fragment has a length of between 50 and 3000 nucleotides.

14. Use according to claim 11 or 12, characterized in that said pigmentation is that of the hair.

15. Use according to claim 11 or 12, characterized in that it relates to the prevention or treatment of canities.

16. Use according to claim 15, characterized in that said canities is an early canities.

17. Use of a polynucleotide fragment according to claim 11 or 12, characterized in that said fragment is associated with a fluorescent, radioactive or enzymatic probe.

18. A method for the diagnosis of a predisposition to premature canities in an individual, comprising the following steps: i) selection of a marker belonging to a gene of human chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6,

5 C5orf7, LOC51308, ETFl, HSPA9B, PCDHAI to PCDHA13, CSFIR,

RPL7, PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and FLJ11715, ii) determination of the alleles of the chosen marker present in a sample of genetic material of said individual. _* 10 19. The method of claim 18 comprising the additional step. >< ^r iii) comparison of the alchemical form of the marker with that of other individuals to establish the diagnosis. 20. The method of claim 19 characterized in that the other individuals are members of the same family as that of the individual to be diagnosed. 21. Use of at least one polynucleotide fragment comprising 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, for cosmetic purposes, in the field of pigmentation, said fragment having a length of between 30 and 5000 nucleotides.

22. Use of at least one polynucleotide fragment comprising 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, 25 NPAT, ATM, AF035326, AF035327 , AF035328, BC029536, FLJ20535, DRD2,

ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650, for the manufacture of a medicament for therapeutic use in the field of pigmentation, said fragment having a length of between 30 and 5000 nucleotides. 30

23. Use according to claim 21 or 22, characterized in that said fragment has a length between 50 and 3000 nucleotides.

24. Use according to claim 21 or 22, characterized in that said pigmentation is that of the hair.

25. Use according to claim 21 or 22, characterized in that it relates to the prevention or treatment of canities.

26. Use according to claim 25, characterized in that said canities is an early canities.

27. Use of a polynucleotide fragment according to claim 21 or 22, characterized in that said fragment is associated with a fluorescent, radioactive or enzymatic probe.

28. A method for the diagnosis of a predisposition to premature canities in an individual, comprising the following steps: i) selection of a marker belonging to a gene of 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, ii) determination of the alleles of the chosen marker present in a sample of genetic material of said individual.

29. The method of claim 28 comprising the additional step iii) comparison of the algebraic form of the marker with that of other individuals, to establish the diagnosis.

30. The method of claim 29 characterized in that the other individuals are members of the same family as that of the individual to be diagnosed.

31. Use for cosmetic purposes, in the field of pigmentation, of a combination of at least two polynucleotide fragments each comprising at least 18 successive nucleotides, the sequence of which is chosen from: • a sequence corresponding to all or part of a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and ALI 33097, and • 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, ETFl, HSPA9B, PCDHAI to PCDHA13, CSFIR, RPL7,

PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and FLJ11715, and • 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, BCO 10946 , TAGLN, PCSK7 and ENS300650, said fragments having a length of between 30 and 5000 nucleotides.

32. Use of a combination of at least two polynucleotide fragments chosen from:

A fragment comprising at least 18 consecutive nucleotides, the sequence of which corresponds to all or part of a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNAlD, ARHGEF3 and AL133097, and

A fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from human chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5όrf7, LOC51308, ETFl, HSPA9B, PCDHAI to PCDHAI 3, CSFIR, RPL7, PDGFRB, TCOFl, AL133039, CD74, RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3,

GALNTIO and FLJ11715, and

• a fragment comprising 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,

BCO 10946, TAGLN, PCSK7 and ENS300650, for the manufacture of a medicament for therapeutic use in the field of pigmentation, said fragments having a length of between 30 and 5000 nucleotides.

33. Method for the diagnosis of a predisposition to premature canities in an individual, comprising the following steps: i) selection of a combination of at least two markers chosen from

. the markers belonging to a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097, and

. markers belonging to a gene from human chromosome 5 chosen from the genes KLHL3, HNRPAO, CDC25C, EGR1, C5orf6, C5orf7, LOC51308, ETFl, HSPA9B, PCDHAI to PCDHA13, CSFIR, RPL7, PDGFRB, TCOFl, ALI 33039, CD74,

RPS14, NDSTl, G3BP, GLRAl, C5orf3, MFAP3, GALNTIO and

FLJ11715, and

. markers belonging to a gene from human chromosome 11 chosen from the genes GUCY1 A2, CUL5, ACAT1, NPAT, ATM,

AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 and ENS300650, ii) determination of the alleles of selected markers present in a sample of genetic material of said individual.

34. The method according to claim 33 comprising the additional step iii) comparison of the algebraic form of the marker with that of other individuals to establish the diagnosis.

35. The method of claim 34 characterized in that the other individuals are members of the same family as that of the individual to be diagnosed.

36. 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 a gene from human chromosome 3 chosen from the genes KIAA1042, CCK, CACNA1D, ARHGEF3 and AL133097, those comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene from 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 and FLJ11715, and those comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of a gene in the human chromosome 11 selected from the genes GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DRD2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 long and ENS6 fragments eur between 30 and 5000 nucleotides.