FR2842104A1 - Use of human polynucleotides for treatment, prevention and diagnosis of pigmentation disorders, especially premature whitening of hair, derived from chromosomes 9 or 6 - Google Patents

Abstract

Cosmetic or therapeutic use, in the field of pigmentation, of at least one polynucleotide fragment (I), of 30-5000 nucleotides (nt) and containing at least 18 consecutive nt that correspond to all or part of specific genes present on human chromosomes 9 or 6. Cosmetic or therapeutic use, in the field of pigmentation, of at least one polynucleotide fragment (I), of 30-5000 nucleotides (nt) and containing at least 18 consecutive nt that correspond to all or part of specific genes present on human chromosomes 9 or 6. The specified genes are: (a) for chromosome 9: FREQ, NT-030046.18, NT-030046.17, GTF3C5, CEL, CELL, FS, ABO, BARLH1, DDX31, GTF3C4 and Q96MA6; and (b) for chromosome 6: HLAG, NT-007592.446, .506, .507 or .508, HSPA1B, G8, NEU1, NG22, BAT8, HLA-DMB or -DMA,BRD2, HLA-DQA1 or -DQA2, NT-007592.588, .459 or .457, GRM4, RNF23 or FLJ22638. Independent claims are also included for the following: (1) diagnosing a predisposition to premature canitis (whitening of the hair) by identifying the alleles of the specified genes; (2) use of at least one of the SNP (single-nucleotide poymorphism) markers 418620, rs302919, 913705, 931886, 429269, 2526008, 2734988, 2734967, 1419664, 2517502, rs733539, rs494620, 154973, 206779, 60071 or rs763028 for determining genes implicated in pigmentation of the skin and other external parts of the body; (3) similar use and method of a combination of at least one (I) derived from chromosome 9 and at least one derived from chromosome 6; (4) kit containing at least two (I); and (5) similar method and use using a combination of at least one (I) derived from DDX31 or GTF3C4 and at least one fragment derived from the following genes (present on chromosomes 3, 5 or 11) KIAA1042, CCK, CACNA1D, ARHGEF3, AL133097, KLHL3, HNRPA0, CDC25C, EGR1, C5orf3, 6 or 7, LOC51308, ETF1, HSPA9B, PCDHA1 to 13, CSF1R, RPL7, PDGFRB, TCOF1, AL133039, CD74, RPS14, NDST1, G3BP, GLRA1, MFAP3, GALNT10, FLJ11715, GUCY1A2, CUL5, ACAT1, NPAT, ATM, AF035326, AF035327, AF035328, BC029536, FLJ20535, DED2, ENS303941, IGSF4, LOC51092, BC010946, TAGLN, PCSK7 or ENS300650. ACTIVITY : Dermatological. No details of tests for dermatological activity are given. MECHANISM OF ACTION : Modulating gene expression or activity of encoded products.

Description

G NES INVOLVED IN CANITIE PR COCE

  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. Of

  this fact, this solution is temporary and must be frequently renewed.

  In this context, the inventors chose to explore the appearance of hair

  whites, 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. It also helps identify the genes that are involved in canities. This identification opens the door to numerous applications, in the field of cosmetics of course, but also for carrying out diagnostic tests. In addition, depigmentation of the hair is sometimes one of the symptoms of an illness and in such. case, it is possible that the diagnosis of canities allows the diagnosis of this disease, or that the treatment of canities has a

  therapeutic effect on said disease.

  The present invention describes for the first time the use of genetics in a cosmetic project. 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 biochemistry 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 genes identified as responsible for premature canities are most likely also involved in aging canities. The second obstacle to 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.

  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 involved with a 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 statistically involved in canities, will be referred to indifferently as "chromosomal regions of the invention" or "genomic regions of the invention" or "chromosomal zones of the invention" or "zones

genomics of the invention ".

  The object of the present invention relates on the one hand to the identified chromosomal regions and on the other hand to the use of derivatives, such as transcription or translation products, in the fields of cosmetics, therapeutics and

of the diagnosis.

  For the fields of therapy and cosmetics, the present invention successively relates to the use of polynucleotides derived from a chromosomal region of the invention, the use of agents capable of modifying the function attached to a region of the invention, the use of the expression products of a region of the invention and the use of agents capable of modifying the function of these expression products. Joint or combined use of at least two of the above products

  can 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 chromosomal regions of the invention. In terms of diagnosis, it may also be particularly relevant to combine the information coming from different 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 sequence 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

  stable Watson Crick bond between the two strands.

  Although the base pairing is preferably of the WatsonCrick type, other types are not excluded, such as a Hoogsteen or Hoogsteen type pairing

1 5 reverse.

  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 else 3. by complementarity, or else

  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 error rate accepted,

  they preferably have a difference in length of 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 about 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 to more than 90%, they correspond to one another. 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 authorized concerning the possibility of introduction, of errors in these processes, that is to say that it is considered that two DNA sequences remain "corresponding" if less globally introduced. one in 10 error in the

  complementarity or identity process, 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 the cleavage of address signals, all the resulting polypeptides are

  also considered as expression products 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 may be that of telomeres and relate to degeneration.

  Other specific functions attributed to DNA fragments are well known by

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 the markers makes it possible to follow

  their transmission within the framework of family trees.

  Among the conventional markers, mention may be made of markers which exhibit a restriction fragment length polymorphism. This type of polymorphism is

  called RFLP (Restriction Fragment Length 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 10 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 sequences

  microsatellites are therefore widely used as genetic markers.

  Normally, there is no explicit link between a microsatellite 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 106

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. Widely used markers are microsatellite markers for 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 terminals 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 the nearest Okb, indicating the microsatellite markers surrounding it.

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

including markers.

  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 pattern of gene transmission

  and a marker in suitable families.

  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 step, a phenotype is associated with the defective or modified form of the gene. An error in the assignment of certain phenotypes falsifies 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

  valid only 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 5 cM binding means that there is a 95% chance of reaching a correct conclusion and only 1 in 20 chance that a

  recombination has occurred between the marker and the disease locus.

  The inventors have identified two distinct chromosomal regions, belonging to chromosomes 6 and 9, which are involved in premature canities. These two regions

  are each of the chromosomal regions or zones of the invention.

  According to a first aspect, the invention relates to the region of human chromosome 6 identified by the inventors as being involved in premature canities and the uses of products derived from this region, such as transcription or expression products. This first chromosomal zone of the invention is delimited on chromosome 6 by the microsatellite markers D6S1629 and D6S257. This area will be more

  particularly called "first chromosomal zone of the invention".

  According to a second aspect, the invention relates to the region of human chromosome 9 identified by the inventors as being involved in premature canities and the uses of products derived from this region, such as transcription or expression products. This second chromosomal area of the invention is delimited on chromosome 9 by the microsatellite marker D9S290 and the telomeric region (long arm telomer). This area will more particularly be called "second or second

  chromosomal area of the invention ".

  For the 2 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 chromosomal zones of the invention, these DNA fragments having the functional characteristic to be involved in the

  canities, or in early 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 area of human chromosome 6 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 this area. This first chromosomal zone of the invention is delimited on chromosome 6 by the microsatellite markers D6S1629 and D6S257. Preferably, the fragments thus defined have a sequence corresponding to all or part of the sequence delimited by the markers D6S1610 and D6S1280, included in the first chromosomal zone of the invention. According to other preferred particular cases, the sequence of the fragments corresponds to all or part of the first chromosomal zone of the invention delimited by the markers D6S1629 and D6S1610, or by the markers D6S1610 and D6S1019, or by the markers D6S1019 and D6S1017, or by the markers D6S1017 and D6S1280, or by the markers D6S1280 and D6S1960, or by

the markers D6S1960 and D6S257.

  In the area delimited on human chromosome 6 by the microsatellite markers D6S 1629 and D6S257 reside genes, some of which are involved in the HLA system. Therefore, at least a portion of the polynucleotide fragment corresponding to all or part of the first chromosomal zone of the invention, can correspond to a part of an HLA gene. In the opposite case, the fragment can correspond, at least in part, to a so-called "non-HLA" gene which specifically

  does not belong to the category of HLA genes.

  The second aspect of the invention relates to the area of human chromosome 9 identified by the inventors as being involved in premature canities. According to this aspect of the invention, a fragment as covered by the invention has a sequence corresponding to all or part of this area. This second chromosomal area of the invention is delimited on chromosome 9 by the microsatellite marker D9S290 and the telomeric region (long arm telomer). Preferably, the fragments thus defined have a sequence corresponding to all or part of the sequence delimited by the markers D9S290 and D9S158, included in the second chromosomal zone of the invention. According to other preferred particular cases, the sequence of the fragments corresponds to all or part of the second chromosomal zone of the invention delimited by the markers D9S290 and D9S164, or by the markers D9S164 and D9S158, or by the marker D9S158 and the region

  telomeric at the end of the long arm of chromosome 9.

  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 chromosomal zone in question. 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 1 00 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 one of the two chromosomal zones of the invention. According to a preferred case, the fragment corresponds more particularly to at least part of a gene, included in one of the two chromosomal zones of the invention. In a particular case of this situation, it is all or part of an exon of a gene included in one of the two zones

chromosomes of the invention.

  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 two chromosomal zones of the invention.

  For these two chromosomal zones, the present invention relates first of all to uses in the field of cosmetics. By cosmetic

  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 may be that of the skin or of the integuments. It can be the color of the pigmentation as well as the absence of pigmentation; quality and intensity issues

  pigmentation are also concerned by 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 one applies to the area to be treated a

  composition comprising at least one product of the invention.

  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 product is applied to the area to be treated. invention. The areas to be treated can be, for example and without any limitation, leather

  hair, eyebrows, mustache and / or beard.

  More particularly, the methods for treating canities and natural pigmentation of gray or white hair and / or body 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 composition completely 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 in accordance with the invention has proved to be particularly advantageous when it is applied in the form of a hair lotion,

  possibly rinsed or even in the form of a shampoo.

  For the two identified chromosomal zones called "chromosomal zones 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 deprived

  of protection against the sun's rays which constitute for them an important danger.

  Other conditions involving pigmentation are also concerned by 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 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, the 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 medicinal products, they may 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 is either 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 cells, or 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 two chromosomal zones 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 or any other molecule envisaged in the definition of fragment

  polynucleotide given above.

  Regarding its environment, this fragment can be or be part of a plasmid, a viral genome or another type of vector. In other cases, it can be part of the genome of a cell, or part of a cell genetically modified to

  include 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 may also be surrounded by Missing 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

  (Chain Reaction polymerization) 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 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

  depending on the characteristic he wants 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, a

  sequencing, microsequencing or a mismatch detection test.

  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 the region of human chromosome 6 between the markers D6S 1629 and D6S257 or to all or part of the region of chromosome 9 human between the marker D9S290 and the telomer of the long arm. In particular, a fragment according to the invention may contain only 18 bases complementary to 18 successive bases from one of the two regions

  chromosomes previously described.

  According to another particular case, the fragment described can be the cDNA or the RNA of one of the two chromosomal regions previously described. It can correspond to one or more exons from one of the regions of the invention, it can correspond to a sequence

  of regulation included in one of the regions identified on chromosomes 6 and 9.

  In the context of this first use, the number of polynucleotide fragments as defined above is not limited and is not restricted

necessarily one.

  In particular, use may be made of several polynucleotide fragments whose sequence corresponds, at least in part, to the first chromosomal zone of the invention. Preferably, the sequences of the different fragments correspond to regions distinct from the first chromosomal zone of the invention, for example

separate exons.

  Alternatively, use may be made of several polynucleotide fragments whose sequence corresponds, at least in part, to the second chromosomal zone of the invention. However, within the framework of this first use, the various polynucleotide fragments which are used all have at least part of their sequence

  corresponding to the same chromosomal region of the invention.

  This first use according to the invention is preferably in the field of

cosmetics.

  This use may 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 one of the chromosomal areas 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, part of the sequence of which corresponds to one of the two chromosomal regions of the invention identified by the inventors, or it may be able to modulate the function of an endogenous sequence included in one of these two chromosomal areas of the invention. Preferably, an agent serving for this second use according to the invention not only modulates the function of a fragment

  of exogenous DNA as defined, but also of the corresponding endogenous DNA fragment.

  The DNA fragment whose function is modulated may partially correspond to the first chromosomal region of the invention. Alternatively, the DNA fragment whose function is modulated can partially correspond to the second chromosomal region of the invention. In particular, it may be a plasmid having only a short sequence corresponding to one of the chromosomal regions mentioned. Preferably, the sequence correspondence is established over at least 18

successive nucleotides.

  All the foregoing remarks, in the definition section, on what is to be understood by "function of a DNA fragment" are valid to consider all of 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 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. The same is true 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 partly to one of

  two chromosomal regions 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 6 identified by the inventors. According to the second aspect of the present invention, the chromosomal region in question is that identified by the

inventors on chromosome 9.

  Agents according to the invention are, for example, single-stranded DNA molecules capable of binding to defined sub-regions of one of the two chromosomal regions of the invention, to form triple helices. Under these conditions, agents according to

  the invention abolishes 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 two chromosomal regions of the invention. Preferably, agents according to the invention are enhancers or inhibitors which bind to regulatory regions of the region of human chromosome 6

  between the markers D6S1629 and D6S257.

  Alternatively, agents according to the invention are enhancers or inhibitors which bind to regulatory regions of the region of human chromosome 9

  between the marker D9S290 and the telomer of the long arm.

  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 two chromosomal regions.

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 bases.

  successive of one of the two chromosomal regions previously described.

  This second use according to the invention is preferably in the field of

cosmetics.

  This use may 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 fixing itself, 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 by attaching itself to a possible inhibitor which will thereby cease

to deactivate 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 two chromosomal regions of the invention. For therapeutic uses, the agent thus defined is used in the manufacture of a medicament. Preferably, the fragment

  of DNA 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 the first chromosomal region of the invention. In 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,

  may correspond at least in part to the second chromosomal region of the invention.

  Such an agent according to the invention may be capable of modulating the function of the expression product of an exogenous DNA fragment, part of the sequence of which corresponds to one of the two chromosomal zones of the invention identified by the inventors , or it may be able to modulate the function of the expression product of an endogenous sequence included in one of these two chromosomal zones of the invention. Preferably, an agent serving for this third use according to the invention not only modulates the function of an expression product of an exogenous DNA fragment such 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 the first chromosomal region of the invention , or the second chromosomal region 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 fixation

normal 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 the first chromosomal region of the invention. These agents may or may not be of a protein 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.

  According to the other aspect of the invention, agents as defined in the present application can modulate the function of the protein encoded by a DNA fragment corresponding at least in part to the second chromosomal region of the invention

identified by the inventors.

  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 does not

  not necessarily restricted 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 same chromosomal region of the invention. According to the first aspect of the invention, this region is that of the human chromosome 6 identified by the inventors. According to the second aspect of the present invention, the chromosomal region in question is that identified by the inventors on chromosome 9. This third use according to the invention is preferably in the field of

cosmetics.

  This use may 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 product.

  expression 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 passage of RNA -protein. Another preferred situation is to choose an agent

  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 two chromosomal regions 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

minus 18 nucleotides.

  In particular, such an expression product is the RNA transcript derived from a DNA fragment corresponding at least in part to the first chromosomal region of the invention, or else the second chromosomal region of the invention, whatever the maturation stage 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. Of

  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 can be an expression product derived from exogenous DNA, at least part of the sequence of which corresponds to part of one of the two chromosomal regions of the invention. The use of an agent which is perfectly synthetic but similar to the product of expression of an exogenous or endogenous DNA fragment corresponding at least in part to one of the two chromosomal regions 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 first region

  chromosome of the invention or to the second chromosomal region 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 complementary to the target fragments normally synthesized by the cell are thus obtained. 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 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 expression products of a DNA fragment whose sequence belongs to or corresponds at least in part to one of the two chromosomal regions of the invention, n ' is not limited and can 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 the same chromosomal region of the invention. According to the first aspect of the invention, this region is that of the human chromosome 6 identified by the inventors. According to the second aspect of the present invention, the chromosomal region in question is that identified by the

inventors on chromosome 9.

  This fourth use according to the invention is preferably in the field of

cosmetics.

  This use may 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 fragment

DNA 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 binding , at

  transcribed 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 uses in cosmetics are preferably in the field of

pigmentation.

  For the four types of use described above, the product of the invention

  may 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 area of the skin) and / or the scalp or 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 into 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 can be presented in all

  dosage forms normally used, particularly in cosmetology.

  A preferred composition of the invention is a suitable cosmetic composition

  topical application to the scalp and / or 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 else 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 treating lotion, a styling cream or gel, a dye composition (in particular oxidation dyes) optionally in the form of shampoos dyes, restructuring lotions for

hair, mask.

  When the invention consists of a 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 considered.

  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, moreover,

contain lipid vesicles.

  When the composition is an oily solution or gel, the fatty phase may

  represent more than 90% of the total weight of the composition.

  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.

  For example, the microspheres can be prepared according to the method

  described in patent application EP 0 375 520.

  The nanospheres may be in the form of an aqueous suspension and 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 0 641 557 and EP 0 705 593).

  The products of the invention may also be encapsulated in nanocapsules consisting of a lamellar coating obtained from a silicone surfactant (see patent application EP 0 780 115), the nanocapsules may also be prepared for

  base of water-dispersible sulfonic polyesters (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 1 016 453,

  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 1 160 005 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 0 447 318 and EP 0 557 489) and containing a cationic surfactant on the surface (see references cited

  previously for cationic surfactants).

  In particular, a composition such as the coating of the

  products of the invention has a diameter less than or equal to 10 gim.

  In known manner, the composition may 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).

  As emulsifiers which can be used, mention may, for example, be made of glycerol stearate, polysorbate 60 and the mixture of PEG-6 / PEG-32 / Glycol Stearate sold under the name

  name of 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, there may be mentioned 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: the agents modulating the differentiation and / or the proliferation and / or the pigmentation of skin cells such as retinol and its esters, vitamin D and its derivatives, estrogens such as estradiol, 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 ca-tocopherol or its esters, superoxide dismutases or its mimetics, certain metal chelators or ascorbic acid and its esters; - anti-seborrhoeics such as certain sulfur-containing amino acids, 13-cis retinoic acid, cyproterone acetate; - other agents for combating desquamative conditions of the scalp such as zinc pyrithione, selenium disulfide, climbazole, undecylenic acid, Ketoconazole, piroctone 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: - esters of nicotinic acid, 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-piperidinopyrimidine 3oxide or "Minoxidil" described in US patents 4, 139,619 and US 4,596,812; agents for inhibiting lipoxygenase or inducing cyclo-oxidase promoting hair regrowth such as those described by the Applicant in European patent application EP 0 648 488; - antibacterial agents such as macrolides, pyranosides and tetracyclines, and in particular Erythromycin; - 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; - Sterodian or non-steroidal 5-cL-reductase inhibitors such as those described by the Applicant in European patent applications EP 0 964 852 and EP 1 068 858 or else finasteride; - ATP-dependent potassium channel agonists such as cromakalim

and nicorandil.

  In another implementation possibility, the present invention relates to

  methods for the diagnosis of 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

  individuals under the age of 18.

  As it is very likely that environmental factors play a role in the "canities" phenotype as in that of "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. The term “marker” is intended to mean a DNA sequence the different allelic variations of which 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.

  According to a first aspect of a method of the invention, the marker or markers selected belong to the region of human chromosome 6 comprised between the microsatellite markers D6S1629 and D6S257, that is to say to the first chromosomal zone of the invention, preferably in the region delimited by the markers

D6S1610 and D6S1280.

  According to a second aspect of a method of the invention, the marker or markers selected belong to the region of human chromosome 9 between the microsatellite marker D9S290 and the telomeric region, that is to say to the second area

chromosome of the invention.

  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 both 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

  coupled 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; tests

  hybridization 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, biallelic markers may prove to be particularly suitable if one allelic form reflects a predisposition to premature canities while the other allelic form on the contrary reflects the absence of such a predisposition. Other more common markers are polymorphic and may be

  found in at least two allelic forms and generally more than two.

  Among the markers which can be selected in the first step of the process of the invention, particularly well known markers are the microsatellite markers. The properties of these markers have already been fully described in the context of the present invention. 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 in which 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 can be very wise to

  select several markers, in order to establish a diagnostic test as reliable as possible.

  When the markers selected are microsatellite markers, the various allelic variants correspond to the numbers of tandem repeats of the motif characterizing the marker. A particularly advantageous situation to look for when selecting the marker corresponds to the situation where certain numbers of repetitions in

  tandem 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 following markers: D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960, D6S257. These markers are microsatellite markers belonging to the first chromosomal zone of the invention. A particularly preferred marker is D6S 1017. 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 following markers: D9S290, D9S164 and D9S158. These markers are microsatellite markers

  belonging to the second chromosomal zone of the invention.

  The methods of the invention are not limited to the two stages described and

  may contain other steps before or after the two steps mentioned.

  In particular, a method of the invention may include the additional step of comparing the allelic form of the selected marker or markers with the allelic 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

  markers in individuals known to be free from such a predisposition.

  Given that premature canities are a presumably multigene disorder, 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, you must

  preferably repeat this operation for all 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 is sought to modulate comprises at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 6 between the markers D6S 1629 and D6S257. 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 the region of human chromosome 9 between the marker D9S290 and the telomer of the arm long. 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 of the process is the detection of a variation of a parameter linked to the function of said fragment, for example the detection of a possible fixation of this molecule on the polynucleotide fragment highlighted.

  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 zones

  chromosomes 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 capable of

  determine what is the parameter whose variation 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

  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 the region of human chromosome 6 between the markers. D6S1629 and D6S257. According to the second 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 the region of human chromosome 9 between the marker D9S290 and the long arm telomer. Preferably, the fragment

  of DNA includes 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 product.

  of expression demonstrated by a ligand detection technique.

  As mentioned previously in the application, the expression product of a DNA fragment is understood to mean both the RNA molecules 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 the folding, before and after cleavage of the different signals

  addressing, 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 whose variation 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

  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 two chromosomal zones of the invention. A particular use envisaged by the present invention therefore consists in using the microsatellite markers described above within each of the chromosomal regions of interest with the aim of locating more finely the genes involved in pigmentation, and more particularly those involved

  in the gradual or sudden interruption of the pigmentation of the skin or the integuments.

  In the context of these uses, according to the first aspect of the invention, the microsatellite markers which are used for the determination of the genes of interest are chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280,

D6S 1960 and D6S257.

  According to the second aspect of the invention, the microsatellite markers which are used for the determination of the genes of interest are chosen from the markers

D9S290, D9S164 and D9S158.

  The present invention is based on the identification by the inventors of two chromosomal zones, on human chromosomes 6 and 9, involved in the phenomenon of pigmentation or depigmentation. This genetic basis enabled them to envisage the uses in therapy and in cosmetics described above, as well as the methods of

  diagnosis illustrated previously.

  But as already mentioned, the inventors suspect that many genes are involved in the phenomena of pigmentation, and in those linked to the regulation and the cessation of this pigmentation. Therefore, an important part of the present invention consists in combining the results obtained for the two chromosomal zones of

  the invention, to obtain the most additional information.

  In particular, a first use of combination 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 the region of chromosome 6 human between the markers D6S1629 and D6S257 or that of the region of human chromosome 9 between the marker D9S290 and

  the telomeric region (long arm telomer).

  Preferably, among the various polynucleotide fragments which are used, at least two have sequences corresponding to two distinct chromosomes. It is also conceivable that for the same chromosomal area of the invention, 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 chromosomal zone, but with the small variations allowed by the definition of "correspondence sequences", that is to say at most one different nucleotide out of 10, preferably 1 out of 100. The fragments according to the invention contain at least 18 successive nucleotides, these 18 nucleotides forming the sequence which must correspond at least partially to

  one of the two chromosomal zones 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 fragments.

  For therapeutic uses, the polynucleotide fragments enter

  in the manufacture of a medicine.

  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 among the fragments belonging and / or corresponding to all or part of the region of human chromosome 6 between the markers D6S1629 and D6S257, and those belonging and / or corresponding to all or part of the region of human chromosome 9

  between the marker D9S290 and the telomeric region (telomer of the long arm).

  Preferably, among the various agents which are used, at least two modulate the function of DNA fragments corresponding to two distinct chromosomes. 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

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 two chromosomal zones of the invention. All the preferred uses of such agents have already been explained in detail

  in the part describing the second use according to the invention.

  For therapeutic uses, the agents are used in the manufacture of a

1 0 medication.

  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 the region of human chromosome 6 between the markers D6S1629 and D6S257, and those belonging and / or corresponding to all or part of the region of

  human chromosome 9 between the marker D9S290 and the telomeric region.

  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. 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 expression product of a DNA fragment or else modulate different expression products of a DNA fragment, for example RNA at different stages of

  maturation, or RNA 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

  two chromosomal zones of the invention.

  All the preferred uses of such agents have already been explained in detail

  in the part 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 the region of human chromosome 6 comprised between the markers D6S1629 and D6S257, and those belonging and / or corresponding to all or part of the region of human chromosome 9 comprised between the

  marker D9S290 and the telomeric region.

  Preferably, among the different expression products which are used, at least

  at least two are from DNA fragments corresponding to two separate chromosomes.

  It is also conceivable 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 RNAs at different stages of maturation, or RNA from different splices. The same possibilities are offered for

  polypeptides resulting from the translation of RNA.

  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

  two chromosomal zones of the invention.

  All of 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 part of the

manufacture of a drug.

  For the four types of combination uses described above in the context of the invention, the uses in cosmetics are preferably in the field of

pigmentation.

  Among the many combinations envisaged by the present invention, a very advantageous combination comprises at least one polynucleotide fragment corresponding to all or part of the region of human chromosome 6 between the

  markers D6S 1610 and D6S 1280 for the first use of combination.

  For the second combination use, among the agents used, preferably at least one of these agents modulates the function of a DNA fragment corresponding to or belonging to the region of human chromosome 6 between the

markers D6S1610 and D6S1280.

  For the third combination use, among the agents used, preferably at least one of these agents modulates the function of the expression product of a DNA fragment corresponding to or belonging to the region of chromosome 6

  human between the markers D6S1610 and D6S1280.

  For the fourth combination use, among the expression products which are used, preferably at least one of these expression products is derived from a DNA fragment corresponding to or belonging to the region of chromosome 6 human

  between the markers D6S1610 and D6S1280.

  In all of the combination uses described above, in cosmetics or therapy, use is made of several fragments or of several expression products of fragment or of several agents modulating the function of fragments, or indeed of several agents modulating the function of fragment expression product, where the fragment in question corresponds to all or part of one of the two chromosomal zones of the invention. Preferably, at least one of the fragments in question corresponds to the region of human chromosome 6 comprised between the markers D6S 1629 and D6S 1610, or to that comprised between the markers D6S1610 and D6S1019, or to that comprised between the markers D6S1019 and D6S1017, or that included between the markers D6S1017 and D6S1280, or that comprised between the markers D6S1280 and D6S1960, or that

  between the markers D6S1960 and D6S257.

  Preferably, at least one of the fragments in question in the combination uses corresponds to the region of human chromosome 9 between the marker D9S290 and the telomeric region (telomer of the long arm), or to that comprised between the markers D9S290 and D9S164, or that between the markers D9S164 and D9S158, or that between the marker D9S158 and the region

telomeric.

  For the four types of combination use described above, the combinations of the invention may be incorporated into a cosmetic composition or

  pharmaceutical as described above.

  In order to take advantage of the combination of these chromosomal regions, the present invention also relates to combination methods. These processes are implemented

  for the determination of 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 6 between the markers D6S1629 and D6S257, the markers belonging to the region of human chromosome 9 comprised between the marker D9S290 and the telomeric region. Preferably, among the markers selected, at least two do not belong to the same chromosomal region of the invention. According to a particular case of the present invention, the markers, at least two in number, are chosen from the following list of markers: D6S1629, D6S1610, D6S1019, D6S1017, D6S1280,

  D6S1960, D6S257, D9S290, D9S164 and D9S158.

  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 originating from the individual who is undergoing the diagnostic test. Two different alleles,

  carried by both versions of the chromosome, can be identified.

  The methods of implementing these processes have already been explained in the

  part devoted to diagnostic procedures.

  The combination methods of the invention are not limited to the use of two markers, nor are they limited to the two steps described and can

  contain other steps before or after the two steps mentioned.

  In particular, a method of combining the invention may include the additional step of comparing the allelic form of the selected markers with the allelic 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 for the diagnostic methods already mentioned in the present application, a particularly interesting situation consists in comparing the alleles of the markers selected with the 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 two chromosomal areas of the invention. A particular use envisaged by the present invention therefore consists in using a combination of the microsatellite markers described above 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 '' progressive or sudden interruption of pigmentation of the skin or integuments. In the context of these uses, according to the invention, the microsatellite markers which are used for the determination of the genes of interest are chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960, D6S257, D9S290, D9S164 and D9S158. The combination used comprises at least two markers and among the markers used, at least two markers correspond to

separate chromosomes.

  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 the region of human chromosome 6 between the markers D6S1629 and D6S257, and those comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the

telomeric region.

  Caption of the figures Figure 1: Composition of the families analyzed for linkage with the candidate region Figure 2: Candidate region for PC on chromosome 6: Chromosome location and distribution of markers Figure 3: Figure 4: Graphical representation of the NPL scores obtained for l global genome multi-point nonparametric linkage analysis on CP families for

chromosomes 6 and 9.

  Abscissa = position on the genetic map (0 = pter) Ordinate = NPL score Schematic representation of the CP loci identified on chromosomes 6 and

9 by the global genome study.

  The distance between markers is indicated in cM.

  Figure 5: Figure 6: Figure 7: Figure 8: Figure 9: Figure 10: Figure 11: Figure 12: Figure 4A: Chromosome 6, locus6p21-p12 Figure 4B: Chromosome 9, locus 9q34

  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 itself according to its score.

  Lod potential scores by family as a function of the heterogeneity rate

genetics of PC.

  Lod scores detailed by family according to the rate of genetic heterogeneity

  of the CP: new simulation after collection of the new families.

  New simulation on families finalized for the search for candidate chromosomal regions. Lod potential scores depending on the rate of heterogeneity.

  The results are expressed by family.

  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.

  Comparison of family composition between region-candidate analysis and Genome-global analysis.

  Lod potential scores according to the heterogeneity rate of the PC.

  The results are expressed by family.

  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.

EXAMPLES

EXAMPLE 1

  Summary of the work carried out In order to locate the gene (s) for premature canities (PC), a program of analysis of segregation (genetic bond) was 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 informative individuals (presenting and not presenting the trait). The study was carried out according to two main approaches, targeted analysis on a candidate region and global genome study on the twenty-two autosomal chromosomes and the X chromosome. Of all the analyzes carried out by fixing or not fixing parameters for the transmission of the CP trait, two potential loci emerge on chromosomes 6 and 9. The locus on chromosome 6p2l-pl2 between the markers D6S1629 and D6S1280, in the region of the genes of the major histocompatibility complex (MHC, HLA) present strong evidence to contain a predisposition gene and another locus

  (chromosome 9q3 1-q32) also shows 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 character of early hair whitening in some.

  To explore canities due to their genetic aspect, a study of DNA segregation has been 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 assigned to only individuals who presented white hair before 25 years of age and whose half of the hair was gray at years. Families were selected for study based on their performance

  statistics in segregation analysis.

  The part of the study carried out is described according to four main periods: A- Period 1: Determination of the potential of the study. A first selection of families

  more informative 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

  link simulation makes it possible to estimate the potential of the 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

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 simulation of link analysis At the end of an attempt to select families with premature canities according to informativeness criteria for gene localization, 29 families were submitted to a genetic linkage analysis simulation. 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 = 3.00). In order to gather the maximum chance of success, it was very important that the diagnosis of PC be assigned

rigorously.

  The result of this study allows, depending on the robustness of the clinical evaluation, to

  qualify the families which are then collected for genetic study.

  1. Criteria for attributing the CP phenotype Twenty-nine families among the 65 were selected 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 combinations

  allelics (genotypes) in each individual.

  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 evaluate

  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

  once an individual is abducted for an uncertain phenotype.

  The family trees have been redesigned using pedigree management software, which also builds coded files (preplink 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 SLINK simulation software, an availability code (cd) (table 1) is assigned. It thus balances, by a code

  from 0 to 3, the informative nature of each individual in the study.

  The phenotype of each individual was assigned based on the information provided.

  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):

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

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

  Table 1: Definition of availability codes availability code (cd) DNA phenotype O unavailable known 1 available unknown 2 available known 3 unavailable unknown Table 2: Definition of phenotypes <25 years 25 years> 25 years no white hair OO 1 some white hair ( qb) (by 50%) 2 O 0 b. Assigning the phenotype according to age In order to avoid the risk of errors, the following criteria have been defined for

  assigning the phenotype as a function of age in individuals under 30 years of age.

  However, during the final clinical examination, it is desirable that the definition of the phenotype

either more quantitative.

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

  allelic) was finally set at 200.

  The line frequency was set at 1%. The number of possible alleles for the

  genotype is set 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 GENORMAX families according to the maximum Lod score (Zmax) achieved (group A-E). Figure 5 reports the Lod score

simulated for each family.

  Table 3: Family statistics / maximum Lod scores. Detailed results can be found in

the table in figure 5.

  Maximum Lod Score (LMx) number of families group Zmax> ou = 4 7 A 3 <Zmax <4 6 B 2 <Zmax <3 6 C 1 <Zmax <2 7 D Zmax <l 3 E The maximum Lod score cannot be only 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 case of a link 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

(Maximum Lod score in bold).

  1- according to the current diagnosis (maximum lod score) locus of the trait distance 0.0 0.01 0.05 0.1 Lod score 2.28 2.24 2.08 1.88 2- 3 individuals incorrectly diagnosed 0.2 1.44 (A2, 0.3 0.95 Rio, R19) distance 0. 0 Lod score -3.89 3- 2 individuals i

0.01 0.05 0.1 0.2

-1.75 -0.90 -0.50 -0.14

  L incorrectly diagnosed (A2, distance 0.0 0.01 0.05 0.1 Lod score -2. 85 -0.75 -0.05 0.22 4- 1 individual incorrectly diagnosed 0.2 0.36 (R19) 0.3

- 0.01

  R19) 0.3 0.30 0.3 0.63 0.4 0.43 0.4 0.01 0.4 0. 17 aaa distance Lod score 0. 0 1.24 0.01 1.24 0.05 1.23 0.1 1.16 0.2 0. 94 0.4 0.27 a 3. Discussion, conclusion and decisions This simulation study allows to place the 29 families preselected in groups according to the maximum potential Lod score. Although a link is significant as soon as the Lod score value 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 achieved. So the probability that the actual Lod score for families in the

  group B (3 <Zmax <4) is quite small.

  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 and even families in 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 make it possible to 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 genetic linkage results.

  The families of groups D and E (Zmax <2) are not very informative for an analysis

genetic link.

  It seems, subject to a robust clinical characterization, that the families of groups A, B and C are suitable for a genetic linkage analysis study and that they must be collected (individuals with cd = 2). In fact, genetic linkage analyzes carried out 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 of 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 period 1, the 19 families (groups A, B, and C) selected to form a bank in which pedigrees will be taken for genetic linkage analyzes, were contacted for confirmation of the PC diagnosis and

  collection of a series of affected and unaffected individuals.

  This phenotype medical verification campaign confirmed many, but not all, of the PC diagnoses. 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 the failure to retain a certain number of

  families and reduces the informativeness potential of several other 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 the verification of the phenotypes, the inventors simulated a linkage 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 approximately

one 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.

  Heterogeneity rate 0%%% Average

5.094620

1. 619190

0.835383

StdDev

1.679698

1.553049

1.022004

minimun

1.221207

0.000000

0.000000

Maximum

8.835722

7.409957

5.517145

Max epoquel

38823

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

again checked.

  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 loci. 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 6: Final list of families 1 c103974

2 F104512

3 CAN33

4 CAN35

CAN43

6 CAN46

7 CAN46B

8 Can53

9 CAN55

CAN62

1i CAN65

12 CAN65B

  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.

  Heterogeneity rate 0%%% Average

4.752321

1.535356

0.719737

StdDev

1.748924

1.418022

0.978920

minimun

0.356854

0.000000

0.000000

Maximum

8.914062

6.078132

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 connection

significant (Table 8).

  Table 8: Probabilities (in%) of reaching or exceeding a Lod score of 1, 2 or 3 for each rate of heterogeneity Lod score Rate of heterogeneity 1,000 2,000 3,000 0%

99.500

95,500

84.500

%

57,000

31,000

14,000

%

27,000

  8.500 3.500 C- Epoch 3: Analysis of genetic linkage of the PC with the candidate region 1- Hypothesis The region 6p21 is called "candidate region" (CR) because it is associated with premature canities through auto-affections immune systems (Biermer's disease, Basedow's disease,

  Thyrodites, Myasthénie) 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 of the

  power of the sample (Table 10).

  Table 9 New simulation on the 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.

  Heterogeneity rate 0%%%% Average

4.367608

1.354325

0.666549

0.222622

StdDev

1.649267

1.359845

0.891330

0.378039

minimun

0.564761

0.000000

0.000000

0.000000

Maximum

9.042465

6.610168

4.988076

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 Lod score 1,000 2,000 3,000

0% 50%

%%

98,000

94.500

79.000

48,500

23,500

12. 000

21,500

  9,500 3,000 4,000 0.500 0,000 3- Microsatellite markers used Distribution of markers on the candidate region

shown in Figure 2.

  of chromosome 6 is 4- Binding analyzes Several types of binding analyzes have been carried out in order to increase the

  probability of observing an existing link between this chromosomal region and the PC.

  For this analysis, the following two approaches were carried out: - 2 points (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 multi-point method.

  2. Independent analysis of the mode of transmission of the trait, nonparametric (NPL): Analysis of deviation in the proportion of shared alleles for each pair of affected individuals (in each family by identity by descent) compared to random transmission. In multi-point analysis, all pairs of hits (all-pairs,

  Z-all = loglo score of p-values, and p-values) were considered. In the 2points method, it is the pairs of germains that have been studied (affected sib-pair, p-values).

- 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, this means that the

  position is intermediate between 2 markers.

  Global 2-point multipoint Chromo- i Marker / - | Marker / Marker / exit Region Lod position cM Lod position cM NPL position cM 6 _ .. p2._ 120 ._. 1 01/5 1.2 5835 6 II 1 6 1 6p2l

1.12 @ 0.1

  D6Sl 017/54 1.28 58 1 3.56 D6SlOl7 / 54 /,., W 0v $ 52-points Itpit <t- 5 Chromo- | o "î <rsa1? Yes ô'W-È | NL | a1 _ so 6 6p2l '35'-: D8S y6093 O 5S4 6- Discussion and conclusion This study distinguishes a predominant locus of predisposition to premature canities on chromosome 6p2l -pl2 in the HLA gene region (around D6S1017; maximum scores NPL = 3.52, p = 0. 000514, HLod: = 2.01). The binding region (with a confidence level of 99%, NPL> 2.50) is located between positions 41 and 67 on the map of selected markers. The non-parametric analysis provides a series of significant values between positions 47 cM and 58 cM (between D6S1019 and D6S1280). The Lod score although of suggestive rank supports also the location of an important gene on

chromosome 6p.

  These results positively document the presumptions of association which carry

  on the region of chromosome 6p2l with respect to premature canities.

  It should be noted that a family (CAN35) shows a relatively high bond.

  The result of the binding on chromosome 6p2l in the HLA region is very encouraging if we remember that the literature provides few examples of such informative nonparametric analyzes. It provides a promising starting point for the identification of genes of susceptibility to Early Canity, using strategies

associations of polymorphisms.

  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 great analysis also allows

  to estimate the rate of genetic heterogeneity of the PC.

  1- Content of families These are the same families as those that were studied in the candidate regions phase (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.

  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 A35 A46 A65 A53 B43 B55 C33 C62 A46B A65B Total individuals Candidate region Genome Global In order to confirm the benefit of the evolution of the sample, a new simulation analysis of binding was carried out for different situations of genetic heterogeneity (Table 13 and tables of 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 (i.e. 50-70% with the use of microsatellite markers showing a

average heterozygosity of 0.7).

  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.

Heterogeneity rate 0%%%%

90%

Way

4.751841

3.115806

1.378378

0.672997

0.242418

StdDev

1.749689

1.866821

1.467071

0.904472

0.402384

minimun

0.334792

0.024841

0.000000

0.000000

0.000000

Maximum

9.338889

8.780452

7.508479

5.226281

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 = 1.0, Z = 2.0, Z = 3.0. The probability of finding a significant connection (Table 14 and Figure 12) with 4/5 of the families linked to 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 tables in Figure 12.

  Heterogeneity rate 1,000 2,000 3,000 0%

99,000

93. 500

84,000

%

87.500

69.500

48,500

%

47,000

25,500

* 15,000

%

23,000

  il.500 3.500% 5.500 0.500 0.000 The analyzes can therefore indicate significant links if the heterogeneity of the sample does not exceed 20% (i.e. only 1/5 of the families not binding to a single major locus), but it it is still possible to identify a link in case the

  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 genotyped for 400 polymorphic markers distributed on the 22 autosomes and the X chromosome (Table 15) according to an average inter-marker interval of 9.2 cM (density ). These are DNA microsatellites, which are composed of tandem repeats of the type

  dinucleotide (CA) n from the Généthon collection (Evry, France).

  Table 15: Number of markers analyzed for each chromosome during the wide-scan genome. Chromosome Nb of markers

1 31

2 30

3 23

4 22

22

6 20

7 22

8 14

9 20

20

11 18

12 19

13 14

14 14

14

16 13

17 15

18 14

19 12

1 3

21 5

22 7

X 18

  total 400 The average heterozygosity rate observed is 0.70, and the average size of the interval

inter-marker is at 9.2 cM.

  3- Linkage analyzes For this global approach, the inventors 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 PC. Parametric analysis, more efficient in terms of power, takes into account

  of the trait transmission mode and is most suitable in the case of monogenic traits.

  Non-parametric analysis which identifies a link even if the mode of

  assumed transmission is erroneous, is also more robust in the case of multigenic 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) ò Transmission mode (dominant), * Line frequency (1%), * Allelic equi-frequencies of all markers, * Defined penetrations (90% mutant heterozygotes - 100% homozygotes mutants),

  * 2-point and multipoint methods.

  * Link probability scores are expressed in: - Lod score Z (homogeneous sample); - ZH Lod score (heterogeneous sample) and ca heterogeneity rate

  (proportion of families which are not linked to this locus).

  b- Independent analysis of the line transmission mode, nonparametric

(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 individuals affected for

  multi-point analysis, and the first pairs for 2-point analysis.

  The link probability scores are expressed in - NPL or Z-all (Logl O 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 a- detailed results For chromosome 6 in particular, the 20 markers belonging to the global genome battery (GG) were first analyzed, then the analysis was repeated by adding the 13 markers used for the candidate-region analysis (RC) with the 20 markers of the

global genome.

  Figure 3 reports the NPL scores obtained for the nonparametric binding analysis on chromosomes 6 and 9.

  Table 16 reports the best results retained for each type of analysis (PL, NPL) discussed below: Table 16: Best results retained for each type of analysis (PL, NPL) on

  markers used for GG analysis except * (* 33 RC + GG markers).

  Chromosome Positionipter Score 2-point (2P) or Multipoint (MP) Nonparametric npl> 3.0 Z-ail 6 57 x 3.9 MP *

9,151 A MP

  npl> 2.0 Z-ail 9 131.00 p 2.13 MP 6 154.1 00001 2, z 2P Parametric i In terms of PL: Lod score interesting (1.0 <ZH <1.5) - chromosome 6p2i-pl2, position 61 (MP- ZH = 1.43) - chromosome 6ql3-ql4, position 90 (2P-ZH = 1.46) ii In terms of NPL: the loglo scores were distinguished here for the study of deviation from allelic sharing, identity by descent (IBD), for all pairs of affected (Z-all multipoint study scores) as well as the p values for the pairs of affected siblings (affected sib-pairs, scores

2-point study, p-values).

  On chromosome 6p21-pl2, the score reached using the genomeglobal markers maximizes to a Z-all = 3.52 at position 71 (between markers D6S1610 and D6S257). However, the precision of the locus is probably affected by the great distance between these 2 markers of the genome-global battery which is more clearly more important.

  than the mean interval observed (26.11 cM).

  Given the size of this interval, an additional analysis was performed on all 33 markers used during the GG and RC analyzes. This analysis makes it possible to find a higher score (Z-all = 3.59) for the position 57cM (see

table 16).

  The second best score is reached on chromosome 9q31-q32, position 151 (Z-all = 3.37) iii The locus which is identified simultaneously by PL and NPL:

PL NPL

  6p21-pl2, position 57-61 1.42 3.59 - Discussion and conclusion 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) have been observed for chromosomes 6p2l-pl2 (NPL MP Zall = 3.59) and 9q31-q32 (NPL

MP Z-all = 3.37).

  Among these 2 loci, the most robust is that of 6p2l-pl2 which is reinforced by an MPPL Lod score, which although average, maximizes at ZH = 1.42.

  D- General discussion and conclusion At the end of the different periods of analysis, several chromosomal regions are identified or suggested. It is the region 6p21-pl2 (see Figure 4A) which has collected the best consensus for a genetic link to the trait CP. The discrepancy between parametric and nonparametric analyzes generates some uncertainty as to the importance of the role of these

  genes in the pathophysiology of canities.

  The second locus is on chromosome 9q31-q32 with an almost NPL score

significant (Z-all = 3.37).

  a Chromosome 6p2l-pl2: The limits of the region designated by the linkage analysis are located at position 41 (upper limit, Z-all = 2.02) and position 95 (lower limit, Z-all = 2.09) either 54 cM. It is possible to reduce this region to a length of 15 cM by considering 65 cM and 80 cM limits (between D6S 1610 and D6S257 respectively; analysis of 20 markers)

  or 50 cM and 73 cM (between D6S1629 and D6S1280 respectively; analysis of 33 markers).

  The region identified contains the genes of the major histocompatibility complex (MHC, HLA). However, it should not be excluded that a gene independent of HLA could be

  involved in steering, or susceptibility to the CP trait.

  b Chromosome 9q34 The inventors place the proximal limit of this region from the position which collects a score Z-all = 2.5 on the marker D9S290; the distal limit being placed on the telomer of the long arm of chromosome 9 (towards the marker D9S158). This region extends

  over a length of 10 cM (Figure 4B).

EXAMPLE 2

  Examples of compositions - hair fragment lotion DNA fragment from the chromosomal zone between the markers D6S1629 and D6S257 0.5 g Propylene glycol 20g Ethanol 950 30 g Water qs 100 g This lotion is applied daily to the areas to be treated and preferably

  on the whole 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 zone between the marker D9S290 and the long arm telomer 1.5 g Polyglyceryl 3-hydroxylarylether 26 g Hydroxy propyl cellulose sold under the name Klucell G by the company Hercules 2 g Preservatives qps Ethanol 95 50 g Water qs 100g 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 repigmentation

progressive gray hair.

  This shampoo can also be used as a preventive measure to delay the

hair whitening.

  - Gel treating DNA fragment from the chromosomal zone between the markers D6S1629 and D6S257 0.75 g Essential oils of Eucalyptus lg Econozole 0.2 g Lauryl polyglyceryl 6 cetearyl glycoether 1.9 g Preservatives qs Carbopol 934P sold by the 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, there is a repigmentation of the hairs

or hair from the treated area.

  References 25. E. Lander and L. Kruglyak. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results [see comments]. Nat.Genet. Il (3): 241-247, 1995.

Claims (59)

claims   1. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 6 between the markers D6S1629 and D6S257, for cosmetic purposes, in the field of pigmentation , said fragment having a length   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 the region of human chromosome 6 between the markers D6S1629 and D6S257, for the manufacture of a medicament for therapeutic use in the area of   pigmentation, said fragment having a length of between 30 and 5000 nucleotides.   3. Use according to claim 1 or 2, characterized in that said fragment corresponds to all or part of the region of human chromosome 6 between the markers D6S1610 and D6SI019.   1 5 4. Use according to claim 1 or 2, characterized in that said fragment corresponds to all or part of the region of human chromosome 6 between the markers D6S1019 and D6SI017.   5. Use according to claim 1 or 2, characterized in that said fragment corresponds to all or part of the region of human chromosome 6 between the markers D6S1017 and D6S1280.   6. Use according to claim 1 or 2, characterized in that at least a portion of said   fragment corresponds to all or part of an HLA gene.   7. Use according to claim 1 or 2, characterized in that said fragment has a   length between 50 and 3000 nucleotides.   8. Use according to claim 1 or 2, characterized in that said pigmentation is that of the appendages.   9. Use according to claim 8, characterized in that said integuments are the hairs and hair system.   10. Use according to claim 8, characterized in that said integuments are the hair.   Request No. 02 09696 11. Use according to claim 1 or 2, characterized in that it relates to prevention or the treatment of canities.   12. Use according to claim 11, characterized in that said canities is an early canities. 13. Use of a polynucleotide fragment according to claim 1 or 2, characterized in   that said fragment is isolated, purified or recombinant.   14. 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.   15. 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 the region of human chromosome 6 between the markers D6S1629 and D6S257, ii) determination of the alleles of the chosen marker present in a sample   genetic material of said individual.   16. Method according to claim 15 characterized in that said marker is a microsatellite marker. 17. Method according to claim 15 characterized in that said marker is chosen from the following markers: D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960, D6S257.   18. Method according to any one of claims 15 to 17, characterized in that the   polymorphism of said marker indicates the presence or absence of a predisposition to premature canities.   19. The method of claim 15 comprising the additional step iii) comparison of the allelic 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 of the markers chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960 and D6S257 for the determination of genes   involved in pigmentation of the skin or integuments.   Request no 02 09696 22. Use of at least one of the markers chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960 and D6S257 for the determination of the genes involved in the gradual or sudden interruption of the pigmentation of the skin or integuments. 23. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the long arm telomer, for cosmetic purposes, in the area of pigmentation, said fragment having a   length between 30 and 5000 nucleotides.   24. Use of at least one polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the long arm telomer, 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.   25. Use according to claim 23 or 24, characterized in that said fragment corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and D9S 158.   26. Use according to claim 23 or 24, characterized in that said fragment corresponds to all or part of the region of human chromosome 9 between the markers D9S290 and D9S 164.   27. Use according to claim 23 or 24, characterized in that said fragment corresponds to all or part of the region of human chromosome 9 between the markers D9S164 and D9SI58.   28. Use according to claim 23 or 24, characterized in that said fragment corresponds to all or part of the region of human chromosome 9 between the   marker D9S 158 and the long arm telomer.   29. Use according to claim 23 or 24, characterized in that at least one portion   of said fragment corresponds to all or part of a gene.   30. Use according to claim 23 or 24, characterized in that said fragment has a   length between 50 and 3000 nucleotides.   31. Use according to claim 23 or 24, characterized in that said pigmentation is that of the appendages.   Request no 02 09696 32. Use according to claim 31 characterized in that said integuments are the hairs and hair system.   33. Use according to claim 31 characterized in that said integuments are the hair. 34. Use according to claim 23 or 24, characterized in that it relates to the   prevention or treatment of canities.   35. Use according to claim 34, characterized in that said canities is an early canities. 36. Use of a polynucleotide fragment according to claim 23 or 24, characterized   in that said fragment is isolated, purified or recombinant.   37. Use of a polynucleotide fragment according to claim 23 or 24, characterized   in that said fragment is associated with a fluorescent, radioactive or enzymatic probe.   38. 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 the region of human chromosome 9 between the marker D9S290 and the telomer of the long arm, ii) determination of the alleles of the chosen marker present in a sample   genetic material of said individual.   39. Method according to claim 38 characterized in that said marker is a marker microsatellite.   40. Method according to claim 38 characterized in that said marker is chosen from among   following markers: D9S290, D9S164 and D9S158.   41. Method according to any one of claims 38 to 40, characterized in that the   polymorphism of said marker indicates the presence or absence of a predisposition to premature canities.   42. The method of claim 38 comprising the additional step iii) comparison of the allelic form of the marker with that of other individuals to establish the diagnosis.   43. The method of claim 42 characterized in that the other individuals are   members of the same family as that of the individual to be diagnosed.   Request No. 02 09696 44. Use of at least one of the markers chosen from markers D9S290, D9S164 and D9S 158 for the determination of the genes involved in the pigmentation of the skin or the integuments. 45. Use of at least one of the markers chosen from markers D9S290, D9S164 and D9S158 for the determination of the genes involved in the progressive interruption or   sudden pigmentation of the skin or integuments.   46. 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 whose sequence is chosen from: has a sequence corresponding to all or part of the region of human chromosome 6 between the markers D6S1629 and D6S257, has a sequence corresponding to all or part of the region of human chromosome 9 between the marker D9S290 and the long arm telomer,   said fragments having a length of between 30 and 5000 nucleotides.   47. Use of a combination of at least two polynucleotide fragments chosen from: * a fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 6 between the markers D6S 1629 and D6S257, 20. a fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the long arm telomer, for the manufacture of a medicament for therapeutic use in the area of pigmentation, said fragments having a length of between 30 and 5000 nucleotides.   48. Use according to claim 46 or 47, characterized in that at least one fragment corresponds to all or part of the region of human chromosome 6 between the markers D6S1610 and D6S1280.   49. Use according to claim 46 or 47, characterized in that at least one fragment corresponds to all or part of the region of human chromosome 6 comprised between the markers D6S1629 and D6S1610, or to that comprised between the markers D6S1610 and D6S1019, or to that comprised between the markers D6S1019 and D6S1017, or to that Application n 02 09696 comprised between the markers D6S1017 and D6S1280, or to that comprised between the markers D6S1280 and D6S1960, or to that comprised between the markers D6S1960 and D6S257.   50. Use according to claim 46 or 47, characterized in that at least one fragment corresponds to all or part of the region of human chromosome 9 comprised between the markers D9S290 and D9S164, or to that comprised between the markers D9S164 and   D9S158 or that between the marker D9S158 and the long arm telomer.   51. A 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 * markers belonging to the region of human chromosome 6 included between the markers D6S 1629 and D6S257, * the markers belonging to the region of human chromosome 9 comprised between the marker D9S290 and the long arm telomer, ii) determination of the alleles of the chosen markers present in a sample   genetic material of said individual.   52. The method according to claim 51 characterized in that said markers are chosen from the following markers: D6S1629, D6S1610, D6S1019, D6S1017, D6S1280,   D6S1960, D6S257, D9S290, D9S164 and D9SI58.   53. The method according to claim 51 comprising the additional step: iii) comparison of the allelic form of the marker with that of other individuals to establish the diagnosis.   54. The method of claim 53 characterized in that the other individuals are   members of the same family as that of the individual to be diagnosed.   55. Use of a combination of markers chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960, D6S257, D9S290, D9S164 and D9S158, for the determination of at least two markers corresponding to distinct chromosomes, genes involved in pigmentation skin or dander.   Request no 02 09696 56. Use of a combination of markers chosen from markers D6S1629, D6S1610, D6S1019, D6S1017, D6S1280, D6S1960, D6S257, D9S290, D9S164 and D9S158, for the determination of the genes involved in the progressive interruption or   sudden pigmentation of the skin or integuments.   57. 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 the region of human chromosome 6 between the markers D6S1629 and D6S257, and those comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the long arm telomer, said fragments having a   length between 30 and 5000 nucleotides.   58. A method of identifying molecules capable of modulating the function of a polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 6 between the markers D6S 1629 and D6S257, comprising the steps of: i) bringing the molecule to be tested into contact with said fragment and ii) detecting a variation in a parameter linked to the function of said fragment,   said fragment having a length of between 30 and 5000 nucleotides.   59. Method for identifying molecules capable of modulating the function of a polynucleotide fragment comprising at least 18 consecutive nucleotides whose sequence corresponds to all or part of the region of human chromosome 9 between the marker D9S290 and the telomer of the long arm , comprising the steps of: i) bringing the molecule to be tested into contact with said fragment and ii) detecting a variation in a parameter linked to the function of said fragment,   said fragment having a length of between 30 and 5000 nucleotides. Request no 02 09696