# Patent

## Title
Hair shape susceptibility gene

## Abstract

A genetic polymorphism and a hair shape susceptibility gene that are related to hair shape, and a method for determining the genetic susceptibility to hair shape in individual test subjects are provided. Disclosed is a hair shape susceptibility gene, which overlaps with a haplotype block in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) of human chromosome 1 and comprises a portion or the entirety of the base sequence of the haplotype block, wherein the haplotype block is determined by a linkage disequilibrium analysis conducted on a single nucleotide polymorphism (SNP) marker whose allele frequency differs statistically significantly between a group having a curly hair trait and a group having a non-curly hair trait, and consists of a base sequence set forth in any one of SEQ ID NO:1 to NO:3.

## Background
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted substitute sequence listing, file name 2537—0680004SequenceListing_ascii.txt; size 267,388 bytes; and date of creation Apr. 4, 2012, filed herewith, is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to a gene related to hair shape, determination of genetic susceptibility to hair shape, detection and/or determination of the type of hair shape, a marker for screening an ingredient effective for the regulation of hair shape, and a use of the marker.
BACKGROUND OF THE INVENTION
The natural shape of human hair is generally classified into straight hair, wavy hair (wave hair), curled hair, and kinky hair (or coiled hair), depending on the degree of curl of the hair. Since the shape of hair and hairstyle constitutes one of the traits that can be easily recognized as physical features of human being, and also serve as an important factor that determines the first impression of a person, the shape of hair and hairstyle is a matter of great interest from a cosmetic viewpoint, irrespective of gender and age. In the case of kinky hair or curled hair with a high degree of curl, the person has trouble that the degree of freedom in hairstyle is limited so that desired styling cannot be achieved. On the other hand, even in the case of straight hair, the person also has trouble that the hair cannot be volumized, and bare skin is easily shown through.
As methods for changing the shape of hair and hairstyle, hairdressing using various hairstyling agents or hair dryers/hair irons, wave/straight permanent treatments, and the like are being extensively carried out. However, although these operations can effectively modify the shape of hair, the operations have no effect on the causative factor that determines the hair shape. These operations, which are the solutions to the above described troubles, are not fundamental solutions but are merely temporary, and in order to maintain the shape of hair and hairstyle, these operations must be repeated frequently. However, on the contrary, these operations cause increased damage to hair, and consequently impair the cosmetic value. For this reason, there is a demand for the development of a method for the intrinsic regulation of hair shape, by which the hair shape can be changed from the beginning of hair growth.
Searching for a causative factor that determines the hair shape and identifying a causative gene thereof are expected to provide useful information in the development of a method for the intrinsic regulation of hair shape. In regard to the factors or genes related to hair shape, there have been reports on the genetic diseases that bring changes to the shape of hair (Non-Patent Documents 1 to 3), acquired kinky hair caused by drugs (Non-Patent Document 4), curly hair model animals (Non-Patent Documents 5 and 6), an the like. However, the factors or genes disclosed in these documents are merely a special example which affect the hair shape, and are not adequate to be considered as causative factors that determine the natural shape of human hair.
Meanwhile, along with the rapid progress in the genome analysis technology in recent years, the correlation between diseases and genes is being gradually clarified. Particularly, not only for so-called genetic diseases that are defined by variation or abnormality of a single gene, but also for polygenic diseases characterized by low penetrance (the ratio of onset of a certain disease in an individual having a variation in a certain gene), such as highly frequent common diseases including lifestyle diseases such as diabetes and hypertension, search for causative genes using non-parametric linkage analysis techniques such as affected sib-pair linkage analysis is frequently carried out (see, for example, Non-Patent Document 7). Further, based on the hypothesis that the variation of a disease-associated gene for a common disease is a highly frequent genetic polymorphism (common variant), and that although the variation is present in healthy persons as well, the prevalence is significantly high inpatients (Common Disease-Common Variant), search for causative genes by means of linkage disequilibrium analysis using a genetic polymorphism (for example, SNP (Single Nucleotide Polymorphism)) is also actively carried out throughout the world (see, for example, Non-Patent Document 8).
More recently, with the progress in the international HapMap Project, a database of general polymorphisms (SNP) of high frequencies such as one million loci or more in four human populations has been established, and research is being conducted on common diseases as well as on general traits in which the phenotype varies with the human race or population, for example, skin color, hair color, and eye color (see, for example, Non-Patent Documents 9 and 10).
Similarly, also in regard to the natural shape of human hair, it can be contemplated that the natural hair shape is a general trait in which the phenotype varies with the human race or population. In general, many Asian people have straight hair, while African people predominantly have kinky hair (or curled hair). Indo-European people have a high ratio of having a trait of wavy hair (wave hair), which is intermediate of the two. The mode of inheritance was first observed by Rostand, J., et al., and they reported that curly hair is an autosomal (semi) dominant trait over straight hair (Non-Patent Document 11). Furthermore, descriptions on the curly hair trait may also be found in the human Mendelian inheritance database of the NCBI (OMIM, http://www.ncbi.nlm.nih.gov/omim/). However, in regard to causative genes that determine the natural shape of human hair, systematic research on genome analysis has not been completed, and no such genes have been found yet.
PRIOR ART DOCUMENTS
Non-Patent Document


    Non-Patent Document 1: Norgett E E et al., Hum. Mol. Genet. 9(18), p. 2761-2766, 2000
    Non-Patent Document 2: Moller L B et al., Hum. Mutat. 26 (2), p. 84-93, 2005
    Non-Patent Document 3: Kjaer K W et al., Am. J. Med. Genet. A. 127A(2), p. 152-157, 2004
    Non-Patent Document 4: Cullen S I et al., Arch. Dermatol. 125(2), p. 252-255, 1989
    Non-Patent Document 5: Du X et al. Genetics. 166(1), p. 331-340, 2004
    Non-Patent Document 6: Mann G B et al., Cell. 73(2), p. 249-61, 1993
    Non-Patent Document 7: Hanis C L et al., Nat. Genet. 13(2), p 161-166, 1996
    Non-Patent Document 8: Altshuler D et al., Nat. Genet. 26(1), p. 76-80, 2000
    Non-Patent Document 9: Sulem P et al., Nat. Genet. 39(12), p. 1443-1452, 2007
    Non-Patent Document 10: Sabeti P C et al., Nature. 449(7164), p. 913-918, 2007
    Non-Patent Document 11: Rostand J et al., “An Atlas of Human Genetics”, Hutchinson Scientific & Technical, London, pp. 26-29, 1964


SUMMARY OF THE INVENTION
The invention provides a hair shape susceptibility gene, which overlaps with a haplotype block in the 1q32.1 to 1832.2 region (D1S249 to D1S2891) of human chromosome 1 and includes a portion or the entirety of the base sequence of the haplotype block, wherein the haplotype block is determined by a linkage disequilibrium analysis conducted on a single nucleotide polymorphism (SNP) marker whose allele frequency differs statistically significantly between a group having a curly hair trait and a group having a non-curly hair trait, and consists of a base sequence set forth in any one of SEQ ID NO:1 to NO:3.
The present invention also provides a hair shape determining marker, which is an oligo- or polynucleotide containing a partial base sequence of the base sequence of the haplotype block described above, or a complementary strand thereof, wherein the partial base sequence consists of a contiguous base sequence containing one or more single nucleotide polymorphisms (SNPs), wherein the SNPs include an SNP whose allele frequency differs statistically significantly between a group having a curly hair trait and a group having a non-curly hair trait, and an SNPs linked to the SNP.
Furthermore, the present invention provides a method for determining the genetic susceptibility of a test subject to hair shape, the method including the following steps (a) to (c):
(a) preparing a genomic DNA derived from a test subject;
(b) detecting, from the genomic DNA, in the haplotype block, a single nucleotide polymorphism (SNP) which exists in the haplotype block described above and whose allele frequency differs statistically significantly between a group having a curly hair trait and a group having a non-curly hair trait, and a single nucleotide polymorphism (SNP) that is linked to the SNP; and
(c) determining, if the allele frequency of the detected relevant SNP is statistically significantly higher in the group of curly hair people than in the group of non-curly hair people, that the test subject has a genetic predisposition to curly hair, and if the allele frequency of the detected SNP is statistically significantly higher in an arbitrary group of non-curly hair people than in the group of curly hair people, that the test subject does not have a genetic predisposition to curly hair.
The present invention also provides a method for determining the genetic susceptibility of a test subject to hair shape, the method including identifying, for any one or more nucleotides of the nucleotide numbers as indicated in the following table that are present in the base sequences set forth in SEQ ID NO: 1 to NO: 3 in the genomic DNA derived from a test subject, whether the nucleotide is nucleotide (i) or nucleotide (ii); and determining, when the nucleotide is nucleotide (i), that the test subject has a predisposition to curly hair, and when the nucleotide is nucleotide (ii), that the test subject does not have a predisposition to curly hair.












TABLE 1









Nucleotide (i)
Nucleotide (ii)




Nucleotide
(having
(no



SEQ ID NO.
Number
predisposition)
predisposition)





















1
1
C
T




1635
A
G




2527
A
G




3766
A
C



2
7519
A
G




16901
G
T




30270
G
A




31333
G
C




50038
A
T




63008
T
G



3
24524
G
T




60701
A
G










Furthermore, the present invention provides a reagent for the determination of the genetic susceptibility of a test subject to hair shape, the reagent including a probe and/or a primer, which hybridizes with the hair shape determining marker of the present invention under stringent conditions.
The present invention also provides a kit for the determination of the genetic susceptibility of a test subject to hair shape, the kit including the reagent described above.
Furthermore, the present invention provides a method for screening a hair shape regulating agent, the method including the following steps (a) and (b):
(a) administering a test substance to a cell containing the hair shape susceptibility gene of the present invention; and
(b) selecting, among the administered test substances, a substance which converts the type of polymorphism of the nucleotide in a marker with a single nucleotide polymorphism (SNP) that is present on the hair shape susceptibility gene or in the vicinity thereof, and the allele frequency of which differs statistically significantly between a group having a curly hair trait and a group having a non-curly hair trait, or a single nucleotide polymorphism (SNP) that is linked to the SNP, to another polymorphisms, as a hair shape regulating agent.
Furthermore, the invention provides a marker for the type of hair shape, consisting of a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a base sequence complementary thereto, or a partial polynucleotide of these polynucleotides, or consisting of a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47, or a partial polypeptide thereof.
The invention also provides a primer for amplifying the marker for the type of hair shape of the present invention, the primer consisting of a partial polynucleotide of a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a base sequence complementary thereto.
Furthermore, the invention also provides a probe for detecting the marker for the type of hair shape of the present invention, the probe consisting of a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a base sequence complementary thereto, or a partial polynucleotide of these polynucleotides.
The invention also provides an antibody for detecting the marker for the type of hair shape of the present invention, the antibody being capable of specifically recognizing a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47, or a partial polypeptide thereof.
Furthermore, the present invention provides a method for detecting and/or determining the type of hair shape, the method including the following steps (a) to (c):
(a) measuring the amount of expression of the marker for the type of hair shape of the invention in a sample derived from a test subject;
(b) comparing the results in the measurement obtained from step (a) with the results of non-curly hair people; and
(c) determining the type of hair shape based on the results obtained from (b).
The invention also provides a method for evaluating or selecting a hair shape regulating agent, the method including the following steps (a) to (d):
(a) contacting a test substance with a cell capable of expressing the hair shape susceptibility gene of the invention or a protein encoded by the gene;
(b) measuring the amount of expression of the gene or the protein in the cell contacted a with test sample;
(c) comparing the amount of expression measured in step (b) with the amount of expression of the gene or the protein in a control cell that has not been contacted with the test substance; and
(d) selecting, based on the results obtained in step (c), a test substance which increases or decreases the amount of expression of the gene or the protein, as a hair shape regulating agent.
The invention also provides a method for evaluating or selecting a hair shape regulating agent, the method including the following steps (a) to (c):
(a) introducing, to a cell capable of expressing the hair shape susceptibility gene for the type of hair shape of the present invention, a fusion gene of the regulatory region of the hair shape susceptibility gene and a reporter gene, and culturing the cell in the presence and in the absence of a test substance;
(b) measuring the amount of expression of reporter gene expression product in the cell culture cultured in the presence of the test substance, and comparing the amount with the amount of expression of an expression product of the reporter gene expression product in the cell culture cultured in the absence of the test substance; and
(c) selecting, based on the comparison results obtained from step (b), a test substance which increases or decreases the amount of expression of the reporter gene expression product, as a hair shape regulating agent.
The invention also provides a method for evaluating or selecting a hair shape regulating agent, the method including the following steps (a) to (c):
(a) contacting a test subject with an aqueous solution, a cell or a cell fraction prepared from the cell, which contains all contain a protein encoded by the hair shape susceptibility gene of the present invention;
(b) measuring the function or activity of the protein in the aqueous solution, cell or cell fraction that has been contacted with the test substance, and comparing the function or activity with that in a control aqueous solution, a control cell or a control cell fraction, which all have not been contacted with the test substance; and
(c) selecting, based on the comparison results obtained from step (b), a test substance which increases or decreases the function or activity of the protein, as a hair shape regulating agent.
The present invention also provides a method for regulating the type of hair shape, the method including controlling the expression of the hair shape susceptibility gene of the present invention in the human hair root area.
According to an embodiment, the hair shape susceptibility gene of the invention is selected from CSRP1, NAV1, IPO9, TMEM58, and NUCKS1.
According to an embodiment of the hair shape determining marker of the present invention, the SNPs include a SNP in a nucleotide selected from the group consisting of the following bases:
(1) in the base sequence set forth in SEQ ID NO:1, nucleotides represented by Nucleotide Numbers 1 (dbSNP database ID: rs576697, T or C), 1635 (rs645390, G or A), 2527 (rs3767542, G or A), and 3766 (rs675508, C or A);
(2) in the base sequence set forth in SEQ ID NO:2, nucleotides represented by Nucleotide Numbers 7519 (rs2271763, G or A), 16901 (rs10920260, T or G), 30270 (rs16849387, A or G), 31333 (rs12127375, C or G), 50038 (rs1495840, T or A), and 63008 (rs10920269, G or T); and
(3) in the base sequence set forth in SEQ ID NO:3, nucleotides represented by Nucleotide Numbers 24524 (rs3805, T or G), and 60701 (rs823114, G or A).
According to another embodiment, the hair shape determining marker consists of a contiguous base sequence having a length of 10 to 601 nucleotides.
According to the embodiment of the reagent of the invention for the determination of the genetic susceptibility of a test subject to hair shape, the probe and/or the primer is hybridized with a region containing SNPs of the nucleotides described in the items (1) to (3) above.
According to an embodiment of the marker for the type of hair shape of the present invention, the partial polynucleotide is a polynucleotide of 15 bases or more.
According to an embodiment of the method of the present invention for detecting and/or determining the type of hair shape, the sample derived from a test subject is an RNA prepared from a biological sample collected from the test subject, or a complementary polynucleotide transcribed from the RNA.
According to another embodiment of the method of the present invention for detecting and/or determining the type of hair shape, the step (a) is a step for contacting a biological sample collected from a test subject with an antibody for detecting the marker for the type of hair shape of the present invention, and measuring the amount of the marker for the type of hair shape of the present invention in the biological sample that has been bound with the antibody.
According to another embodiment of the method of the present invention for detecting and/or determining the type of hair shape, the biological sample collected from the test subject is derived from an epithelial tissue or epithelial cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a set of images of the phenotypes of hair shape;
FIG. 2 is a diagram showing microsatellite markers and the maximum LODs obtained by an affected sib-pair linkage analysis on chromosome 1;
FIG. 3 is a diagram showing microsatellite markers and the maximum LODs obtained by an affected sib-pair linkage analysis on chromosome 11;
FIG. 4 is a diagram showing microsatellite markers and the maximum LODs obtained by an affected sib-pair linkage analysis on chromosome 1;
FIG. 5 is a conceptual diagram of a 3,926-bp haplotype block represented by a base sequence set forth in SEQ ID NO:1, which contains SNP: rs576697 and extends from SNP: rs576697 to SNP: rs12403361;
FIG. 6 is a conceptual diagram of a 76,945-bp haplotype block represented by a base sequence set forth in SEQ ID NO:2, which contains SNP: rs1495840 and extends from SNP: rs2820290 to SNP: rs2250377;
FIG. 7 is a conceptual diagram of a 68,637-bp haplotype block represented by a base sequence set forth in SEQ ID NO:3, which contains SNP: rs823114 and extends from SNP: rs823103 to SNP: rs1772150;
FIG. 8-1 is a graph showing the amounts of expression of the hair shape susceptibility gene in the scalp hair roots of a curly hair group and a straight hair group, A: CSRP1 gene, B: IPO9 gene;
FIG. 8-2 is a graph showing the amounts of expression of the hair shape susceptibility gene in the scalp hair roots of a curly hair group and a straight hair group, C: NUCKS1 gene;
FIG. 9 is a set of photographs showing the images of hair follicle tissue of various human races, while the arrows indicate curved regions;
FIG. 10 is a set of photographs showing the changes in the shape of a hair follicle during culturing in a human hair follicle organ culture system; and
FIG. 11 is a graph showing the effect of a hair shape susceptibility gene expression regulating agent on the hair follicle shape, A: centipeda minima, B: round cardamon.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the provision of a genetic polymorphism and a hair shape susceptibility gene that are related to the natural shape of human hair such as curly hair or straight hair, and the provision of a method for determining the genetic susceptibility of individual test subjects to hair shape based on this information. Furthermore, the present invention relates to the provision of a reagent and a reagent kit, which are useful for conveniently carrying out the method. In addition, the present invention relates to the provision of a marker (polynucleotide or polypeptide) for detecting and determining the natural shape of human hair such as curly hair or straight hair, and to the use of the marker, such as the detection and/or determination of the type of hair shape, or the evaluation and selection of a ingredient effective for the regulation of hair shape using the marker.
The inventors of the invention set a goal of finding a causative gene that determines the natural shape of human hair, and conducted a genome analysis directed to Japanese family lines having curly hair, a group of Japanese curly hair people, and a group of Japanese non-curly hair people. As a result, the inventors identified genetic polymorphisms related to hair shape, that is, hair shape susceptibility SNP markers, and also identified hair shape susceptibility genes in the 1q32.1 to 1q32.2 region of chromosome 1. The inventors of the present invention also investigated the relations between hair shape and the gene expression of various genes in the hair root area, and found that the amount of expression of the hair shape susceptibility genes in the hair root area differs significantly between non-curly hair people and curly hair people. These genes are hair shape susceptibility genes, and can serve as markers for detecting and/or determining the type of hair shape. Based on these findings, the inventors of the present invention finally completed the present invention.
According to the present invention, a hair shape susceptibility gene related to the natural shape of human hair such as curly hair or straight hair, a hair shape susceptibility SNP marker, and a hair shape determining marker utilizing these are provided. When the hair shape susceptibility gene, the SNP marker, and the hair shape determining marker of the present invention are analyzed in detail, research on the mechanism of the hair formation related to the hair shape, and application research such as the development of an adequate method for promoting the regulation of hair shape are made available.
According to the method for determining the genetic susceptibility to hair shape of a test subject, search for a gene that serves as a main factor that determines the hair shape of individual test subjects, and determination of the susceptibility of individual test subjects to the acquired changes of hair shape, that is, the degree of risk of the future change in the hair shape, can be more conveniently and rapidly carried out. Furthermore, based on the results, an adequate method for regulating the hair shape for individual persons can be provided. Further, the determination method can be carried out more conveniently and rapidly, by the reagent for the determination of genetic susceptibility of a test subject to hair shape of the present invention and the kit including the reagent.
According to the present invention, the shape or nature of hair such as curly hair or kinky hair can be detected and determined without damaging the hair. Furthermore, a substance selected according to the method of the present invention for screening an ingredient effective for the regulation of hair shape can be used as a hair shape regulating agent that is effective for the regulation of hair shape, and can also be used for the preparation of a pharmaceutical product, a quasi-drugs, cosmetic materials, health foods and the like, which all contain the agent. Further, according to the present invention, a method for regulating the hair shape using the hair shape susceptibility SNP marker obtained by the present invention can be provided.
1. DEFINITIONS OF TERMS USED IN PRESENT INVENTION
The indication of base sequences (nucleotide sequences), nucleic acids and the like by means of abbreviations in the present specification is as recommended by the specifications of IUPAC-IUB (IUPAC-IUB Communication on Biological Nomenclature (Eur. J. Biochem. 138, 9, 1984), “Guidelines for the preparation of specifications containing base sequences or amino acid sequences” (edited by the Japanese Patent Office), and the symbols conventionally used in the art.
The term “DNA” as used in the present specification encompasses not only a double-strand DNA, but also single-strand DNAs such as a sense strand, and an anti-sense strand constituting the double-strand DNA. Unless particularly stated otherwise, the term “gene” as used herein encompasses all of a double-stranded DNA including human genome DNA, a single-stranded DNA (sense strand) and a single-stranded DNA having a sequence complementary to the sense strand (anti-sense strand), and fragments thereof. Unless particularly stated otherwise, the term “gene” as used herein is intended to indicate any of a regulatory region, a coding region, an exon and an intron without discrimination. Further, the “gene” or “DNA” encompasses a “gene” or “DNA” represented by a specific base sequence, as well as a “gene” or “DNA” which encodes a homologue, a derivative or a variant of a protein encoded by the “gene” or “DNA” represented by a specific base sequence, provided that they have a biological function equivalent to that of the protein.
Furthermore, according to the present invention, the terms “nucleotide”, “oligonucleotide” and “polynucleotide” have the same meanings as nucleic acid, and they are intended to encompass both DNA and RNA. The DNA encompasses all of cDNA, genomic DNA and synthetic DNA. The RNA encompasses all of total RNA, mRNA, rRNA and synthetic RNA. Further, the “nucleotide”, “oligonucleotide” and “polynucleotide” may be double-stranded or single-stranded, and in the case of a “nucleotide” (or an “oligonucleotide” or “polynucleotide”) having a certain sequence, unless particularly stated otherwise, the “nucleotide” is intended to collectively mean “nucleotide” (or an “oligonucleotide” or “polynucleotide”) having a sequence complementary to the sequence. Furthermore, when the “nucleotide” (or “oligonucleotide” or “polynucleotide”) is RNA, the nucleotide symbol “T” indicated in the base sequence may be replaced with “U”.
The term “polynucleotide having a complementary base sequence” means a polynucleotide that is in a complementary relation in terms of nucleotide (i.e., complementary strand or anti-sense strand), to a polynucleotide having an arbitrary base sequence (sense strand). A complementary base sequence encompasses a sequence that is completely complementary to the subject base sequence, as well as a base sequence that can be hybridized with the subject base sequence under stringent conditions. Here, the stringent conditions may conventionally refer to washing conditions of approximately “1×SSC, 0.1% SDS, 37° C.”, and more stringent hybridization conditions may be approximately “0.5×SSC, 0.1% SDS, 42° C.”, and even more stringent hybridization conditions may be approximately “0.1×SSC, 0.1% SDS, 65° C.”. Furthermore, a person having ordinary skill in the art can determine stringent hybridization conditions according to general textbooks (for example, Sambrook, J. & Russell, D., 2001, Molecular Cloning: a Laboratory Manual, 3rd edition, Cold Spring Harbor, N.Y.: cold Spring Harbor Laboratory). An example of a base sequence that can be hybridized with a subject base sequence under stringent conditions may be a base sequence having a homology of 90% or higher, and preferably 95% or higher, with the subject base sequence.
The term “protein” or “polypeptide” encompasses a “protein” or “polypeptide” represented by a specific base sequence or amino acid sequence, as well as a fragment, a homologue, a derivative and a variant thereof, provided that they all have a biological function equivalent to that of the “protein” or “polypeptide”. Meanwhile, the variant encompasses a naturally occurring allele variant, a variant that does not occur naturally, and a variant having an amino acid sequence modified by artificial deletion, substitution, addition and insertion. In addition, examples of the variant include those having a homology in the amino acid sequence of 80% or higher, preferably 90% or higher, more preferably 95% or higher, and even more preferably 98% or higher, with a protein or polypeptide having no variation.
According to the present specification, the homology of amino acid sequences and base sequences is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, the homology is calculated by performing an analysis using a homology analysis (Search homology) program in the genetic information processing software Genetyx-Win (Software Development Co., Ltd.), and by setting the parameter, Unit size to compare (ktup), at 2.
The term “antibody” encompasses a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, and portions of the antibodies described above, which have antigen-binding properties, such as Fab fragments, and fragments produced by a Fab expression library.
In regard to the term “genetic polymorphism” as used herein, when there are two or more genetically determined alleles, the term refers to such an allele gene. Specifically, in a human population, when variations such as substitution, deletion, insertion, dislocation, and inversion of one or plural nucleotides exist at a specific region in the genome of one or plural individuals, with respect to the genomic sequence of one certain individual, the variation is called “genetic polymorphism” if it is statistically ensured that the variation is not a mutation occurring in the one or plural individuals, or if it can be genetically demonstrated that the variation is not a specific variation in the individuals but occurs in the population at a frequency of 1% or greater. Examples of the “genetic polymorphism” as used herein include substitution of one nucleotide with another nucleotide, that is, a single nucleotide polymorphism (SNP); deletion or insertion of one to several tens of nucleotides (DIP); a region includes repetition units of sequence consisting of 2 to several tens of nucleotides as one unit, where the number of the repetition is different (when the unit repeated in the consist of 2 to 4 nucleotides, it is referred to as a microsatellite polymorphism, and when the unit repeated in the region consists of several to several tens of nucleotides, it is referred to as a VNTR (Variable Number of Tandem Repeat); and the like.
The term “hair shape” as used herein refers to the tendency of the overall shape of hair in the head area, which attributes to the shape of individual hairs, such as straight hair, wavy hair or wave hair, curled hair, or kinky hair or coiled hair.
The term “curly hair” as used herein is, unless particularly stated otherwise, a term which collectively refers to the shape other than straight hair in the case of contrasting with straight hair. Therefore, according to the present specification, in the case of contrasting with the “curly hair”, unless particularly stated otherwise, the “straight hair” and the “non-curly hair” are considered to have the same meaning. The “curly hair”, “non-curly hair” and “straight hair” are of relative nature, and can be defined by various methods that will be described below. The “curly hair trait”, “non-curly hair trait”, and “straight hair trait” refer to the phenotypes representing the “curly hair”, “non-curly hair” and “straight hair”, respectively.
The term “hair shape susceptibility gene” as used herein refers to a causative gene that determines the hair shape which is a polygenic trait, and the term “hair shape susceptibility SNP marker” refers to the nucleotide at a site which represents an SNP associated with the trait of hair shape of the individual.
According to the present specification, the terms “genetic susceptibility to hair shape”, “hair shape determining marker” and “marker for the type of hair shape” respectively refer to the genetic predisposition related to the specific hair shape possessed by an individual, and a marker for determining the predisposition.
The term “Affected Sib-Pair Linkage Analysis” as used herein refers to one technique for estimating the location of a target gene (e.g., disease susceptibility gene or the like) using linkage, and is a representative analysis technique for non-parametric linkage analysis which does not assume any mode of inheritance (e.g., autosomal dominant inheritance, recessive heredity, sex-linked gene, or the like) or the penetrance. In the affected sib-pair linkage analysis, family lines including sibs (e.g., brothers and sisters) that are affected (or have a particular trait) are collected, calculation of the likelihood is carried out on the basis of the data obtained by observation of these family lines, and the genetic locus regions of the marker linked to the disease (or the particular trait) are narrowed down. In the case of a group of general (i.e., not affected, or not having a particular trait) sibs, in one genetic locus, a child receives one of the two alleles of one parent (even if the one parent is a homozygote, the alleles are considered to be different from each other). Therefore, in this case, there exist a case in which the sibs receive the same allele, and a case in which the sibs receive different alleles. Since each of the two alleles of a child originates one allele from each of the parents, when the question of how many identical alleles sibs will receive from their parents is considered, there are three cases such as 0, 1 and 2. These three cases are said to have an IBD (Identity By Descent) of 0, 1 and 2, respectively. When a number of sib-pairs are considered, the numbers of the pairs having an IBD=0, the pairs having an IBD=1, and the pairs having an IBD=2 should be counted, and the proportion of the numbers constitutes a certain proportion (1:2:1) according to the probability laws. On the contrary, when sibs that are affected (or have a particular trait) are collected, and the same investigation is carried out with this group, if an observed marker gene is linked to the disease (or the particular trait), this ratio (1:2:1) is deviated (i.e., the number of the pairs having an IBD=2 increases, and the number of the pairs having an IBD=0 decreases). In addition, for a marker gene which is not linked to a gene that is related to the disease (or the particular trait), it can be considered that the ratio has the same distribution (1:2:1) as any arbitrary sibs. In the affected sib-pair linkage analysis, the likelihood of observation data is calculated by utilizing this hypothesis, by taking the difference of the ratio of shared alleles in affected sib-pairs as an index. The likelihood is represented by the following formula:




  
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        j
        =
        1
      
      N
    
    ⁢
    
      
    
    ⁢
    
      
        ∑
        
          i
          =
          0
        
        2
      
      ⁢
      ZiWij
    
  




wherein Wij represents the probability that the affected sib-pair of the jth family line has an IBD=i. The variable is Z=(Z0, Z1, Z2), and the degree of freedom is 2 (Z2=1−Z1−Z0, there are only two independent variables of Z0 and Z1). The ratio with the likelihood in the case where a marker gene and a gene associated with a disease (or a particular trait) are not linked (that is, Z0=0.25, Z1=0.5, Z2=0.25) is taken, and the value of Z which gives the maximum likelihood is determined by the likelihood maximization method (maximum likelihood estimation).
The term “gene frequency” as used herein refers to the proportion occupied by the allele at a genetic locus among the total number of genes present in a group.
The term “haplotype” as used herein means a combination of genetic variations existing in one allele (haploid).
The term “linkage disequilibrium analysis” or “haplotype analysis” as used herein means an analysis of the degree of the intensity of linkage disequilibrium in a genomic region.
The term “linkage disequilibrium” as used herein refers to a phenomenon in the population genetics, in which a non-random correlation is observed in a group between alleles or genetic markers (polymorphisms) at plural genetic loci, that is, the frequency of such a particular combination (haplotype) is significantly increased. They are generally on the same chromosome and constitute genetic linkage, but there are occasions in which even if the alleles are linked, linkage disequilibrium is not observed. Further, in some exceptional cases, linkage disequilibrium may be seen over different chromosomes. For example, when a genetic locus X has alleles a and b (these exist at the same frequency), and a neighboring genetic locus Y has alleles c and d (these exist at the same frequency), the haplotype ac, which is a combination of the respective genetic polymorphisms, is expected to exist at a frequency of 0.25 in the group. When the frequency of the haplotype ac is higher than such an expected value, that is, when a specific genotype denoted as ac appears frequently, it is said that the allele ac is in linkage disequilibrium. Linkage disequilibrium is occurred as a result that the time of natural selection or introduction into a group of a particular combination of alleles is evolutionarily recent, and may be occurred as a result that linked alleles have not reached equilibrium. Therefore, the mode of linkage disequilibrium varies with different groups, such as nations or races, and even in the case where the allele ac in a certain group is in linkage disequilibrium, there are occasions in which the allele ad is in a relation of linkage disequilibrium in other groups. The detection of genetic polymorphism in the linkage disequilibrium is effective in detecting the susceptibility to a disease, regardless of whether the polymorphism itself directly causes the disease. For example, in regard to an allele a of a certain genetic locus X, although the allele is not a causative genetic factor of a disease, the allele may exhibit susceptibility to a disease through the linkage disequilibrium with an allele c of a genetic locus Y.
The “haplotype block” as used herein is defined as a region that is categorized as a genome region for which most of the historical recombination has not been acknowledged, and includes strong linkage disequilibrium. Identification of a haplotype block can be appropriately achieved by those having ordinary skill in the art based on the strength of the linkage disequilibrium, but for example, the identification can be carried out according to the report of Gabriel, et al. (Gabriel, S. B., et al., Science, 296 (5576), p. 2225-2229, 2002). The term “strong linkage disequilibrium” as used herein means the state in which the upper limit of the 95% confidence interval of the linkage disequilibrium coefficient D′, which is calculated in a linkage disequilibrium analysis, exceeds 0.98, and the lower limit is higher than 0.7. The phrase “there is an evidence of strong historical recombination” means a state in which the upper limit of the 95% confidence interval of the linkage disequilibrium coefficient D′ is lower than 0.9.
The term “minor allele” as used herein means an allele having a low gene frequency when two alleles exist in one genetic locus.
According to the present specification, the terms “gene frequency” and “allele frequency” are used for the same meaning, and are terms meaning the proportion occupied by a particular allele in an arbitrary group of genes.
The phrase “statistically significantly different” as used herein means a state in which when a test is carried out according to any statistical technique, the risk (p value) is less than 0.1%, preferably less than 0.07%, even more preferably less than 0.05%, and still more preferably less than 0.01%.
2. IDENTIFICATION OF HAIR SHAPE SUSCEPTIBILITY GENE AND HAIR SHAPE SUSCEPTIBILITY SNP MARKER
Search and identification of a causative gene that determines the natural shape of human hair, which is a multifactorial general trait (hair shape susceptibility gene), can be carried out by a genetic statistical analysis using a technique for trait mapping. That is, SNP(s) that are in the linkage disequilibrium state with the hair shape susceptibility gene can be effectively selected through the identification of curly hair trait loci by an affected sib-pair linkage analysis and case-control association analysis on the curly hair trait loci, and a gene present in a haplotype block containing the SNP(s) can be identified as a hair shape susceptibility gene.
The identification of the hair shape susceptibility gene and the hair shape susceptibility SNP marker of the present invention can be carried out, as will be described specifically in Examples below, by performing an identification method having the following steps:
(i) a step of defining hair shapes, and collecting curly hair family lines, people having a curly hair trait (case), and people having a straight hair trait (control);
(ii) a step of performing an affected sib-pair linkage analysis directed to the entire genome using samples derived from the curly hair family lines, and identifying a curly hair trait locus;
(iii) a step of selecting plural SNP markers which are not unevenly distributed over the entire region in the curly hair trait locus identified in step (ii);
(iv) a step of performing typing of the SNP markers selected in step (iii) using case-derived and control-derived samples, comparing the results of the typing through a statistical processing, and identifying a SNP marker that is recognized to have a significant difference, as a hair shape susceptibility SNP marker;
(v) a step of determining, with regard to the hair shape susceptibility SNP marker, a region where linkage disequilibrium is recognized within an object candidate region and a hair shape susceptibility SNP marker is contained (haplotype block), using the HapMap PHASE data of the International HapMap Project Database, and thereby identifying a hair shape susceptibility gene; and
(vi) a step of determining, for the haplotype extracted from the haplotype block specified in step (v), a SNP locus that is linked with the hair shape susceptibility SNP marker locus determined in step (iv) using the HapMap PHASE data of the International HapMap Project Database, and additionally identifying the SNP thus-determined as an additional hair shape susceptibility SNP marker.
The step (i) is a step of defining hair shapes (curly hair or straight hair) and collecting analysis objects for trait mapping. In regard to the trait mapping, it is necessary to handle the subject trait quantitatively to a certain extent, and thus, the operation of defining hair shape, by which the objects are defined to have a curly hair trait or a straight hair trait, constitutes an important step when the trait mapping is carried out. There are a variety of human hair shapes, and the method for measurement thereof and the method for classification or defining are also various. For instance, examples of the method of defining hair shapes include a method of binarizing the hair shape, in such a manner that curly hair=1 and straight hair=0; a method of measuring the degree of curly hair by any method and quantifying the degree; and a method that is well known to those having ordinary skill in the art (for example, see Japanese Patent Application Laid-Open (JP-A) No. 2005-350801, JP-A No. 2008-268229, Japanese Patent No. 4159515, and the like), but the method is not limited to these. As a more specific example of the method of defining hair shapes, there may be mentioned a method of classifying hair shapes into several grades (for example, 2 to 10 grades, preferably 3 to 8 grades, and more preferably 5 to 7 grades) based on the features such as the overall shape, the degree of curl of the hair (radius of curl), the frequency of the appearance of curl, and/or the synchrony of curl with the groups of hair in the surroundings; and defining, in regard to such classifications, a hair shape having a tendency of a small radius of curl, such as kinky hair and curled hair or strongly wavy hair, as a curly hair trait, and defining a hair shape having a tendency of a large radius of curl, such as wavy hair, almost straight hair or slightly wavy hair, or straight hair, as a straight hair trait.
The step (ii) is a step of carrying out an affected sib-pair linkage analysis on the entire genome using samples derived from a curly hair family line. The constituent members of the curly hair family line for carrying out the affected sib-pair linkage analysis are sibs (a pair among brothers and sisters, two people) determined to have the curly hair trait by the step (i). More preferably, the constituent members consist of a family of 4 people (or 3 people) including the parents of the sibs, and other brothers and sisters (irrespective of the hair shape) or grandparents may also be further added. Furthermore, the number of the curly hair family lines needed to carry out the affected sib-pair linkage analysis can be determined by estimating and/or observing the frequency in the population of the curly hair trait, the frequency of the causative gene (allele frequency), the sib relative risk, or the like, and calculating the number by through simulation. However, the number of the curly hair family line needed is generally 50 family lines to several hundred family lines.
The genetic marker used in the affected sib-pair linkage analysis is not particularly limited as long as it is a genetic polymorphism, but a microsatellite that exists uniformly in the genome and has a large number of alleles is used with preference. A kit for amplifying and detecting a microsatellite (linkage mapping set) is commercially available from Applied Biosystems Corp. (ABI). Meanwhile, in the present invention, ABI PRISM Linkage Mapping Set-MD 10 v2.5 (manufactured by ABI) which covers human chromosome at an average interval of 9.2 cM, and ABI PRISM Linkage Mapping Set-HD 5 v2.5 (manufactured by ABI) which covers human chromosome at an average interval of 5 cM were used.
Furthermore, the microsatellite that serves as a genetic marker can be arbitrarily selected, and can be retrieved from the Comprehensive Human Genetic Maps of the Mammalian Genotyping Service (http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp), NCBI (http://www.ncbi.nlm.nih.gov/) and the like. In this case, it is preferable to select a microsatellite which exists in the genome at an interval of 0.1 to several cM, and has many alleles and high heterozygosity. Furthermore, microsatellite markers can be added to a chromosome in which linkage has been recognized, and the linkage region can be narrowed (detailed mapping). Meanwhile, for the PCR primer for amplifying and detecting the microsatellites that have been arbitrarily selected and added, the base sequence can be retrieved from the NCBI (http://www.ncbi.nlm.nih/gov/), and the primer can be produced based on the retrieved sequence according to an ordinary method using, for example, a commercially available nucleotide synthesizer. At this time, it is preferable to label the probe with a radioactive substance, a fluorescent substance, a chemiluminescent substance, an enzyme or the like so that the detection of the amplification product can be achieved rapidly and easily.
In the affected sib-pair linkage analysis, PCR is carried out using a genomic DNA derived from a curly hair family line as a template, and using a linkage mapping set (ABI) or an amplification primer of a microsatellite marker arbitrarily selected, and thus an amplification product (fragment) is detected. The operations of PCR and the detection of the amplification product can be carried out according to ordinary methods. At this time, when various amplification primers are labeled with different fluorescent dyes (for example, any dyes emitting different fluorescent light, such as 6-FAM (blue), VIC (green), or NED (yellow)), even if amplification products having an identical size are obtained, plural amplification primers can be rapidly detected by separately discriminating the various fluorescent colors.
A statistical test of the linkage can be carried out using commercially available or publicly disclosed genetic statistic software programs which are capable of non-parametric analysis (for example, Genehunter, Linkage Package, Mapmaker/sibs, and the like).
The determination of the region where linkage is recognized was based on the criteria for obtaining a false positive linkage, according to the guidelines provided by Lander and Kruglyak (Nat. Genet., 11 (3), 241-247, 1995) shown below. The guidelines by Lander and Kruglyak (linkage analysis over the entire genome with a multifactorial disease) has come to be actively carried out, but in the linkage analysis of individual genes, the determination of whether the gene function can be causative is also added. However, since the gene function is not taken into consideration in that stage in the analysis of the entire genome, determination criteria (threshold) of significance purely in terms of mathematical genetics are required. Thus, they provided criteria for significance of linkage as shown in the following Table 2 according to simulations.










TABLE 2









Suggestive Linkage
P < 7.4 × 10−4



(Criteria for obtaining a result of one false
LOD > 2.2



positive linkage from the entire genome)



Significant Linkage
P < 2.2 × 10−5



(Criteria for obtaining a result of 0.05 false
LOD > 3.6



positive linkages from the entire genome)



High Significant Linkage
P < 3.0 × 10−7



(Criteria for obtaining a result of 0.01 false
LOD > 5.4



positive linkages from the entire genome)










Through this process, the whole chromosome can be screened, and a region on the chromosome where linkage with the curly hair trait is recognized can be detected. Through further detailed mapping, a specific region on the chromosome can be identified as a curly hair trait locus. The region identified as such is a region where the presence of a hair shape susceptibility gene is strongly suggested.
The step (iii) is a step of selecting, in the curly hair trait locus region identified in the step (ii), plural SNP markers which are not unevenly distributed over the entire region. The SNP markers can be selected by using various databases related to SNP, such as the dbSNP database (http://www.ncbi.nim.nih.gov/SNP/) and the JSNP database (http://snp.ims.u-tokyo.ac.jp/index_ja.html).
Upon the selection of the SNP marker, a SNP which is useful for the identification of a hair shape susceptibility gene is selected. Specifically, in a Japanese group, a SNP having a gene frequency of minor allele of 10% or greater, and more preferably 15% or greater, is selected. When a SNP having such a gene frequency is used, a SNP marker having high reliability can be selected.
In addition, when a SNP marker is selected by using the gene frequency as an index, there are occasions in which the SNP marker is unevenly distributed in a specific narrow region. In this case, if all of the selected SNP markers are used in the identification of a hair shape susceptibility gene, the experiment becomes complicated, and it is also not very effective that SNPs which are neighboring with each other are in the state of linkage disequilibrium. Therefore, it is preferable to select and use SNP markers which are present at a certain interval from one another. As such, when uneven distribution of markers is eliminated by providing a certain interval between them, a comprehensive association analysis can be carried out over the entire object candidate region, and the identification of the hair shape susceptibility gene can be easily carried out. The distance between adjacent SNP markers that are selected as such is preferably 5 kb or greater, and more preferably 5 kb to 10 kb. If this distance is too long, there is a possibility that a region may occur where the extent of the strength of mutual linkage disequilibrium between SNP markers cannot be checked. Furthermore, if this distance is too short, there are so many SNPs for which strong mutual linkage disequilibrium is recognized, and therefore, it is not efficient.
In the comprehensive selection of SNP markers over the entire object candidate region, apart from this distance between SNP markers, the state of scattering of markers in the object candidate region, that is, the number of markers per unit distance of genome, can be expressed as “marker density.” The marker density is 0.5 SNPs or more, preferably 1 SNP or more, and more preferably 1 SNP to 2 SNPs, per 10 kb of genome. If the marker density is too low, the distance between markers is too long, and there is a possibility that a region may occur where the degree of the strength of linkage disequilibrium between SNP markers cannot be checked, as described above. On the other hand, if the marker density is too high, the distance between markers is too short, and as described above, markers are selected overcrowdedly, so that in the case of identifying a hair shape susceptibility gene, a large amount of experiment is needed, which is not so efficient.
The step (iv) is a step of carrying out a case-control association analysis for the SNP markers selected in step (iii). The case-control association analysis is a method of comparing the allele frequencies for a certain hereditary marker between a case (affected people: people having the curly hair trait) group and a control (control people: people having the straight hair trait), and detecting a marker which can exhibit a significant difference in the allele frequency between the two groups. For example, samples derived from people having the curly hair trait (case) and people having the straight hair trait (control) are used, and typing is carried out. The results are compared by statistical processing, and a SNP marker with which a significant difference is recognized is identified as a hair shape susceptibility SNP marker. The sample required for trait mapping is not particularly limited as long as the sample contains genomic DNA, but examples include blood such as peripheral blood, body fluids such as saliva and sweat, somatic cells, and tissues or organs including somatic cells. The number of case and control required to perform a case-control association analysis can be estimated based on the frequency in a population having the curly hair trait, the gene frequency (allele frequency) causative of the trait, the genotype relative risk, and the like, but the number is generally 50 to several thousand people. Furthermore, it is possible to obtain a relatively high power of test by a stepwise refinement method under the conditions of limited sample size, limited number of typing operations or the like. Furthermore, the case and the control are preferably constituted of the same human race as the race for which the hair shape susceptibility gene is specified, and for example, in order to identify a hair shape susceptibility gene of Japanese people, it is preferable that the object of analysis be constituted of Japanese people.
As the method for SNP typing, methods that are well known to those having ordinary skill in the art, such as PCR-SSCP, PCR-RLFP, PCR-SSO, PCR-ASP, a direct sequencing method, SNaPshot, dHPLC, a Sniper method, and a MALDI-TOF/MS method, can be used (see, for example, Nojima, Hiroshi, Ed., “Forefront of Genomic Drug Discovery”, p. 44-p. 54, Yodosha Co., Ltd., 2001). For example, it is effective to utilize TaqMan SNP Genotyping Assays (registered trademark) (manufactured by ABI), and to employ a SNP typing method which utilizes a TaqMan system.
The association analysis is typically achieved by comparing the gene frequency of each of the SNP markers between the case group and the control group, and carrying out a χ2 test on whether the difference in the frequency is statistically meaningful or not (see, University of Tokyo, College of Arts and Sciences, Department of Social Sciences, Statistics Section, Edited, “Tokeigaku Nyumon—Kisotokeigaku I (Introduction to Statistics—Fundamental Statistics I)”, University of Tokyo Press, 1991). However, the association analysis may also be carried out based on the genotype frequency for each SNP marker, the genotype frequency in the case of employing a dominant (or recessive) model, the frequency of allele in terms of positive ratio, and the like. Furthermore, in addition to the χ2 test, the association analysis can be carried out by any other well-known statistical processing, as long as it is possible to compare the case group and the control group, that is, to test the relations between a phenotype that can be divided into plural groups, such as a trait, a disease, and a genetic polymorphism.
Meanwhile, in order to evaluate the typing error of a genotype, and the validity of sampling, a Hardy-Weinberg equilibrium test is carried out. Hardy-Weinberg equilibrium is well known in the field of genome statistics, and in which two alleles (for example, C and T) exists as in an SNP or the like, and the respective frequencies in a group are represented by p and q(p+q=1), the genotype frequencies of C/C homo, C/T hetero and T/T homo may be represented by p2, 2pq and q2, respectively (p2+2pq+q2=1). When an association analysis is carried out, it is desirable that the Hardy-Weinberg equilibrium is established for the control group. However, the selected SNP marker can be evaluated as valid, as long as the number of alleles, whose genotype frequency is statistically significantly different from Hardy-Weinberg equilibrium, is in a predictable range of the significance level (typically, p=0.01 to 0.05).
According to an embodiment, typing is carried out for the respective samples obtained from a case group and a control group, and a significant difference test is carried out by a χ2 test by four methods involving the genotype, allele type, dominance model and recessive model. That is, if a certain genetic variation is causative of hair shape change, the difference in the allele frequency or the like between the case and the control can be predicted. In regard to the test, when the association analysis is carried out on a relatively small number of objects, or when the power of test of the significant difference between the objects is increased, the level of significance is set loose. When the number of objects is relatively large, or when the significant difference is strictly determined, the level of significance can be set strict. A SNP which exhibits a significant difference in the gene frequency by a test is identified as a hair shape susceptibility SNP marker.
The step (v) that is subsequently carried out is a step of determining a hair shape susceptibility gene by specifying, in connection with the hair shape susceptibility SNP marker determined as described above, a region where linkage disequilibrium is recognized in an object candidate region and the hair shape susceptibility SNP marker is included (haplotype block), using the HapMap PHASE data of the International HapMap Project Database.
The analysis of haplotype (linkage disequilibrium analysis) is a method well known to those having ordinary skill in the art, and can be carried out by various linkage disequilibrium analyses that are conventionally carried out (for example, Kamatani, Naoyuki, Edited., “Post-Genome Jidai no Iden Tokeigaku (Genetic Statistics in Post-Genomic Era)”, p. 183-201, Yodosha Co., Ltd., 2002). The haplotype analysis can be carried out using various genetic statistics software programs that are commercially available or made public (for example, Haploview, Arlequin, SNP disease-associated analysis software, SNPalyze (registered trademark) (manufactured by Dynacom Co., Ltd.), and the like). More specifically, the linkage disequilibrium coefficient D′ (pair-wise LD coefficient) is calculated and an analysis is carried out, through a linkage disequilibrium analysis based on the EM algorithm (Laird, N.: “The EM Algorithm”, Chap. 14, pp. 509-520, Handbook of Statistics, Vol. 9, Computational Statistics, C. R. Rao (ed.), Elsevier Science Publishers B.V., 1993). More specifically, in the haplotype analysis, it is analyzed whether linkage disequilibrium exists between the hair shape susceptibility SNP marker specified above and another SNP marker, and the region where linkage disequilibrium exists is identified as the haplotype block. The other SNP marker used in the linkage disequilibrium analysis can be freely selected among the SNPs existing in the upstream and the downstream of the genome sequence with respect to the hair shape susceptibility SNP marker. For example, the linkage disequilibrium analysis may be sequentially carried out for the SNPs present from proximal positions to distal positions of the hair shape susceptibility SNP marker, or the linkage disequilibrium analysis may be carried out for arbitrarily selected SNPs at distal positions to determine an approximate haplotype block region, and then be carried out for SNPs at more proximal positions to determine a more specific haplotype block region. The number of the other SNP markers used in the linkage disequilibrium analysis is 4 SNPs or more including the hair shape susceptibility SNP marker, preferably 20 SNPs or more, and even more preferably 32 SNPs or more, and the analysis is carried out for a series of SNP marker groups including these plural SNP markers. Here, the linkage disequilibrium coefficient D′ is obtained from the following equation when, in two SNPs, the respective alleles of a first SNP are designated as (A, a), the respective alleles of a second SNP are designated as (B, b), and the respective frequencies of four haplotypes (AB, Ab, aB, ab) are designated as PAB, PAb, PaB, and Pab. Furthermore, Min[(PAB+PaB) (PaB+Pab), (PAB+PAb) (PAb+Pab)] in the equation means that the smaller value between the values of (PAB+PaB) (PaB+Pab) and (PAB+PAb) (PAb+Pab) is taken.

D′=(PABPab−PAbPaB)/Min[(PAB+PaB)(PaB+Pab),(PAB+PAb)(PAb+Pab)]

The number of markers in the SNP marker group may appropriately vary with the size of the region forming the haplotype block related to the hair shape susceptibility gene to be identified (linkage disequilibrium block). Furthermore, when a discontinuity of block can be predicted in advance, it is also possible to carry out the analysis on about 6 SNPs located over the blocks. Furthermore, it is also acceptable to carry out a linkage disequilibrium analysis for a hair shape susceptibility SNP marker and 5 SNPs each existing on both sides of the hair shape susceptibility SNP marker 11 SNPs in total. If necessary, the number of markers to be analyzed may be increased.
As the linkage disequilibrium analysis is carried out, a region where SNPs are linked within an object candidate region (a haplotype block including the group of SNP markers among which strong linkage disequilibrium is recognized) is determined. For example, the linkage disequilibrium coefficient D′ is calculated for all combinations between 2 SNPs for the selected SNP markers, combinations showing the relation: D′>0.9 are selected, and a series of regions including a region sandwiched between the remotest SNPs among them are detected. Subsequently, D′ is calculated between three consecutive SNPs that are adjacent to the region in the outside of the detected region, and the SNPs in the region. Even among any combinations thus calculated, when it is verified that D′ is 0.9 or less, the region is specified as a “haplotype block.”
When a haplotype block is determined in this manner, for example, in connection with that region, genes present in the haplotype block under attention can be determined using a database associated with the genome, or the like. Furthermore, even in the case of not using a database, the base sequence in the vicinity of SNP markers present in the haplotype block region are determined by ordinary methods, and genes can also be determined from the base sequence.
The step (vi) is a step of determining, for the haplotype extracted from the haplotype block specified in the step (v), a SNP locus that is linked to the locus of the hair shape susceptibility SNP marker identified in the step (iv) using the HapMap PHASE data of the International HapMap Project Database, and additionally identifying the SNP thus-determined as an additional hair shape susceptibility SNP marker.
In the step (v), it is possible to extract all haplotypes consisting of the respective nucleotides of the SNP marker group used in the haplotype analysis, while simultaneously determining the haplotype block, and to thereby determine the frequency of the haplotype or the like.
When the combinations of the respective nucleotides of the extracted haplotype, that is, the SNP marker group, are compared, a SNP locus that is linked to the locus of the hair shape susceptibility SNP marker identified in the step (iv) can be identified, and the SNP locus thus identified can be designated as an additional hair shape susceptibility SNP marker.
Through the steps (i) to (vi), a chromosome region where linkage with curly hair is recognized is determined, and then a hair shape susceptibility SNP marker is selected from the chromosome region. Furthermore, through a haplotype analysis of the selected SNP marker, a haplotype block and gene in the chromosome region that are related to hair shape are identified. Thereafter, a SNP locus that is linked to the locus of the hair shape susceptibility SNP marker is further determined, and thereby, a hair shape susceptibility SNP marker that is present in the haplotype block or gene can be identified.
Examples of the chromosome region where linkage to curly hair is recognized, which is determined in the steps described above, include chromosome 1 and chromosome 11, more specifically the 1q32.1 to 1q32.2 region of chromosome 1 (a region sandwiched between microsatellites D1S249 and D1S2891) (maximum LOD score=2.14). These regions are determined as curly hair trait loci, and it is strongly suggested that hair shape susceptibility genes exist in these regions.
Examples of the haplotype block specified by the steps described above include, among the genomic regions of human chromosome 1, a 3,926-bp region represented by the base sequence set forth in SEQ ID NO:1, a 76,945-bp region represented by the base sequence set forth in SEQ ID NO:2, and a 68,637-bp region represented by the base sequence set forth in SEQ ID NO:3.
A gene which overlaps with such a haplotype block, and contains a portion or the entirety of the base sequence of the haplotype block, is identified as a hair shape susceptibility gene. Here, the “gene which overlaps with the haplotype block” means both a gene which has the same base sequence as that of a partial region of the haplotype block, and a gene which has the same base sequence as the base sequence of the entire region of the haplotype block. Further, a single nucleotide polymorphism (SNP) which exists in such a haplotype block, and whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, and an SNP that is linked to the SNP, are identified as hair shape susceptibility SNP markers.
An example of the gene which overlaps with the 3,926-bp haplotype block represented by the base sequence set forth in SEQ ID NO:1, may be CSRP1 gene on human chromosome 1. CSRP1 is a gene represented by GeneID:1465 in the Entrez Gene Database (http://www.ncbi.nlm.nih.gov/gene), and as illustrated in Example 5 and FIG. 5, a portion of the base sequence overlaps with the haplotype block described above.
Examples of the hair shape susceptibility SNP marker present in the base sequence set forth in SEQ ID NO:1 include nucleotides represented by Nucleotide Numbers 1 (dbSNP Database ID:rs576697, T or C), 1635 (rs645390, G or A), 2527 (rs3767542, G or A), and 3766 (rs675508, C or A). A preferred example is a nucleotide represented by Nucleotide Number 1 (rs576697, T or C).
Examples of the gene which overlaps with the 76,945-bp haplotype block represented by the base sequence set forth in SEQ ID NO:2 include NAV1 gene, IPO9 gene, and TMEM58 gene on human chromosome 1. NAV1 gene is a gene represented by GeneID:89796 in the Entrez Gene Database, and as illustrated in Example 5 and FIG. 6, a portion of the base sequence overlaps with the haplotype block described above. Further, IPO9 gene is a gene represented by GeneID:55705 in the Entrez Gene Database, and as illustrated in Example 5 and FIG. 6, the entire length of the base sequence overlaps with the haplotype block described above. TMEM58 gene is a gene represented by GeneID:149345 in the Entrez Gene Database, and as illustrated in Example 5 and FIG. 6, a portion of the base sequence overlaps with the haplotype block described above.
Examples of the hair shape susceptibility SNP marker present in the base sequence set forth in SEQ ID NO:2 include nucleotides represented b Nucleotide Numbers 7519 (rs2271763, G or A), 16901 (rs10920260, T or G), 30270 (rs16849387, A or G), 31333 (rs12127375, C or G), 50038 (rs1495840, T or A), and 63008 (rs10920269, G or T). A preferred example may be a nucleotide represented by Nucleotide Number 50038 (rs1495840, T or A).
Examples of the gene which overlaps with the 68,637-bp haplotype block represented by the base sequence set forth in SEQ ID NO:3 include NUCKS1 gene on human chromosome 1. NUCKS1 gene is a gene represented by GeneID: 64710 in the Entrez Gene Database, and as illustrated in Example 5 and FIG. 7, the entire length of the base sequence overlaps with the haplotype block described above.
Examples of the hair shape susceptibility SNP marker present in the base sequence set forth in SEQ ID NO:3 include nucleotides represented by Nucleotide Numbers 24524 (rs3805, T or G), and 60701 (rs823114, G or A). Preferred examples include a nucleotide represented by Nucleotide Number 60701 (rs823114, G or A).
3. HAIR SHAPE DETERMINING MARKER
The present invention also provides a hair shape determining marker in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) of human chromosome 1, which is an oligo- or polynucleotide, or a complementary strand thereof, wherein the oligo- or polynucleotide contains a partial base sequence of the base sequence of a haplotype block that is determined by a linkage disequilibrium analysis for a SNP marker whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait and consists of a base sequence set forth in any one of SEQ ID NO:1 to NO:3, and wherein the partial base sequence consists of a contiguous base sequence containing one or more single nucleotide polymorphisms (SNPs) wherein the SNPs include an SNP whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, and an SNP linked to the SNP.
The oligo- or polynucleotides, or complementary strands thereof, defined by these base sequences contain one or more a hair shape susceptibility SNP marker that is a single nucleotide polymorphism (SNP) which is present in a haplotype block, represented by a base sequence set forth in any one of SEQ ID NO:1 to NO:3, and whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, or an SNP linked to the SNP. When these oligo- or polynucleotides, or complementary strands thereof, are detected, the genetic predisposition of hair shape in a test subject can be examined and/or determined. Therefore, these oligo- or polynucleotides, or complementary strand thereof can be defined and used as markers for determining the genetic predisposition of hair shape possessed by an individual.
The length (nucleotide length) of these oligo- or polynucleotides, or complementary strands, is desirably a length which is specifically recognized in human genome, and there are no particular limitations on the limit. The length is usually equal to or more than 10-mers and equal to or fewer than 1000-mers, preferably equal to or more than 20-mers and equal to or fewer than 500-mers, and more preferably equal to or more than 20-mers and equal to or fewer than 100-mers. Therefore, if necessary, the length can be set to, for example, 11 nucleotides containing a hair shape susceptibility SNP marker present in a haplotype block represented by a base sequence set forth in SEQ ID NO:1 to NO:3 (preferably including 5 nucleotides each on the 5′ side and the 3′ side of the hair shape susceptibility SNP marker), 21 nucleotides (preferably including 10 nucleotides each on the 5′ side and the 3′ side of the hair shape susceptibility SNP marker), 101 nucleotides (preferably including 50 nucleotides each on the 5′ side and the 3′ side of the hair shape susceptibility SNP marker), 601 nucleotides (preferably including 300 nucleotides each on the 5′ side and the 3′ side of the hair shape susceptibility SNP marker), or the like.
Examples of the hair shape susceptibility SNP marker used in the present invention, which should be included in the hair shape determining marker of the present invention, include the following:
(1) nucleotides represented by Nucleotide Numbers 1 (dbSNP Database ID:rs576697, T or C), 1635 (rs645390, G or A), 2527 (rs3767542, G or A), and 3766 (rs675508, C or A) in the base sequence set forth in SEQ ID NO:1;
(2) nucleotides represented by Nucleotide Numbers 7519 (rs2271763, G or A), 16901 (rs10920260, T or G), 30270 (rs16849387, A or G), 31333 (rs12127375, C or G), 50038 (rs1495840, T or A), and 63008 (rs10920269, G or T) in the base sequence set forth in SEQ ID NO:2; and
(3) nucleotides represented by Nucleotide Numbers 24524 (rs3805, T or G), and 60701 (rs823114, G or A) in the base sequence set forth in SEQ ID NO:3.
Among the nucleotides described above, the nucleotide represented by Nucleotide Number 1 (rs576697, T or C) in the base sequence set forth in SEQ ID NO:1, the nucleotide represented by Nucleotide Number 50038 (rs1495840, T or A) in the base sequence set forth in SEQ ID NO:2, and the nucleotide represented by Nucleotide Number 60701 (rs823114, G or A) in the base sequence set forth in SEQ ID NO:3 are preferred.
It is desirable that the hair shape susceptibility SNP marker be located at the center or near the center of the hair shape determining marker of the present invention (for example, within 100 nucleotides, preferably 50 nucleotides, more preferably 30 nucleotides, even more preferably 10 nucleotides, and still more preferably 5 nucleotides, from the center), but it is not necessarily required. Furthermore, when two or more hair shape susceptibility SNP markers are included in the hair shape determining marker of the present invention, all of the hair shape susceptibility SNP markers may be located at the center or near the center of the hair shape determining marker of the present invention; one of the hair shape susceptibility SNP markers is located at the center or near the center, while the others may be located at any positions; or all of the hair shape susceptibility SNP markers may not be located at the center or near the center.
Specific examples of the hair shape determining marker of the invention in which the hair shape susceptibility SNP marker is located at the center include, for example, in the case where a SNP is contained in the nucleotide represented by Nucleotide Number 1 (dbSNP Database ID:rs576697, T or C) in the base sequence set forth in SEQ ID NO:1, a 11-mer polynucleotide consisting of from 5 nucleotides upstream of SEQ ID NO:1 to Nucleotide Number 6, a 21-mer polynucleotide consisting of from 10 nucleotides upstream of SEQ ID NO:1 to Nucleotide Number 11, a 101-mer polynucleotide consisting of from 50 nucleotides upstream of SEQ ID NO: 1 to Nucleotide Number 51, and a 601-mer polynucleotide having a base sequence consisting of from 300 nucleotides upstream of SEQ ID NO:1 to Nucleotide Number 11. Furthermore, complementary strands of these can also be used. In the same manner, the base sequences of markers containing other SNPs are also determined.
4. METHOD FOR DETERMINING GENETIC SUSCEPTIBILITY TO HAIR SHAPE
The present invention also provides a method for determining the genetic susceptibility (genetic predisposition) of a test subject to hair shape. The method for determining the genetic susceptibility to hair shape of the present invention includes the following steps (a) and (b), and there are no particular limitations on the limit:
(a) a step of preparing a genomic DNA derived from a test subject; and
(b) a step of detecting, from the genomic DNA, a single nucleotide polymorphism (SNP) whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, and being present in a haplotype block in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) of human chromosome 1 that is determined by a linkage disequilibrium analysis on a single nucleotide polymorphism (SNP) marker whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, and that consists of a base sequence set forth in any one of SEQ ID NO:1 to NO:3, and a single nucleotide polymorphism (SNP) linked to the SNP.
The step (a) (extraction of a genomic DNA) and the step (b) (detection of SNPs) can be carried out using a known method (for example, Birren Bruce et al., Genome Analysis, Vol. 4/A Laboratory Manual Mapping Genomes, Cold Spring Harbor Laboratory, NY, 1999).
In the step (a), the genomic DNA derived from a test subject can be obtained from a material such as all cells (including cultured cells; however, reproductive cells are excluded), tissues (including cultured tissues), organs, or body fluids (for example, blood, saliva, lymph fluid, respiratory tract mucosa, semen, sweat, urine, and the like), which have been isolated from the test subject, clinical specimens therefrom, and the like. The material is preferably leukocytes or monocytes separated from peripheral blood, and is more suitably leukocytes. These materials can be isolated according to those methods usually used in clinical tests.
For example, in the case of using leukocytes as the material, first, leukocytes are separated from the peripheral blood isolated from a test subject, according to an ordinary method. Subsequently, Proteinase K and sodium dodecyl sulfate (SDS) are added to the leukocytes thus obtained to degrade and denature proteins, and then phenol/chloroform extraction is carried out to thereby obtain genomic DNA (including RNA). The RNA can be eliminated with an RNase as necessary. Meanwhile, the extraction of genomic DNA is not limited to the method described above, and can be carried out using a method well-known in the art (for example, Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual (3 Vol. set), Cold Spring Harbor Laboratory, NY, 2001) or using a commercially available DNA extraction kit or the like. Furthermore, if necessary, the DNA containing the 1q32.1 to 1q32.2 region of human chromosome 1, or a DNA containing a haplotype block represented by a base sequence set forth in any one of SEQ ID NO:1 to NO:3 in the genomic region of human chromosome 1, may be isolated. The isolation of the DNA can be carried out by PCR using a primer which hybridizes with the 1q32.1 to 1q.32.2 region or with the corresponding haplotype block and using the genomic DNA as a template, or the like.
In the step (b), detected from the genomic DNA obtained in the step (a) is an SNP which is a polymorphism present in a haplotype block in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) of human chromosome 1 that is determined by a linkage disequilibrium analysis on a single nucleotide polymorphism (SNP) marker whose allele frequency is statistically different between a group having a curly hair trait and a group having a non-curly hair trait, and the allele frequency of which SNP is higher in any curly hair people group than in any non-curly hair people group, or a SNP that is linked to the SNP. The base sequences set forth in SEQ ID NO:1 to NO:3 include the 3,926-bp base sequence set forth in SEQ ID NO:1, the 76,945-bp base sequence set forth in SEQ ID NO:2, and the 68,637-bp base sequence set forth in SEQ ID NO: 3, in the genomic region of human chromosome 1.
The method for determination of the present invention preferably further includes the following step (c):
(c) a step of determining, if the allele frequency of the detected SNP is statistically significantly higher in the curly hair people group than in the non-curly hair people group, that the test subject has a genetic predisposition to curly hair, and if the allele frequency of the detected SNP is statistically significantly higher in any non-curly hair people group than in the curly hair people group, that the test subject does not have a genetic predisposition to curly hair.
An example of the step (c) may be a step of identifying, for any one or more nucleotides of the nucleotide numbers as indicated in the following table that are present in the base sequences set forth in SEQ ID NO:1 to NO:3 in the genomic DNA derived from a test subject, whether the nucleotide is nucleotide (i) or nucleotide (ii); and determining, when the nucleotide is nucleotide (i), that the test subject has a predisposition to curly hair, and when the nucleotide is nucleotide (ii), that the test subject does not have a predisposition to curly hair.












TABLE 3









Nucleotide (i)
Nucleotide (ii)




Nucleotide
(having
(No



SEQ ID NO.
Number
predisposition)
predisposition)





















1
1
C
T




1635
A
G




2527
A
G




3766
A
C



2
7519
A
G




16901
G
T




30270
G
A




31333
G
C




50038
A
T




63008
T
G



3
24524
G
T




60701
A
G










More specifically, the method of the present invention for determining genetic susceptibility of a test subject to hair shape includes any one step of the following (1) to (12).
(1) In the base sequence set forth in SEQ ID NO:1, it is identified whether the nucleotide represented by Nucleotide Number 1 is T or C, and it is determined, when the nucleotide is C, that the test subject has a predisposition to curly hair, or when the nucleotide is T, the test subject does not have a predisposition to curly hair;
(2) in the base sequence set forth in SEQ ID NO:1, it is identified whether the nucleotide represented by Nucleotide Number 1635 is G or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is G, the test subject does not have a predisposition to curly hair;
(3) in the base sequence set forth in SEQ ID NO:1, it is identified whether the nucleotide represented by Nucleotide Number 2527 is G or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is G, the test subject does not have a predisposition to curly hair;
(4) in the base sequence set forth in SEQ ID NO:1, it is identified whether the nucleotide represented by Nucleotide Number 3766 is C or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is C, the test subject does not have a predisposition to curly hair;
(5) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 7519 is G or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is G, the test subject does not have a predisposition to curly hair;
(6) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 16901 is T or G, and it is determined, when the nucleotide is G, that the test subject has a predisposition to curly hair, or when the nucleotide is T, the test subject does not have a predisposition to curly hair;
(7) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 30270 is A or G, and it is determined, when the nucleotide is G, that the test subject has a predisposition to curly hair, or when the nucleotide is A, the test subject does not have a predisposition to curly hair;
(8) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 31333 is C or G, and it is determined, when the nucleotide is G, that the test subject has a predisposition to curly hair, or when the nucleotide is C, the test subject does not have a predisposition to curly hair;
(9) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 50038 is T or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is T, the test subject does not have a predisposition to curly hair;
(10) in the base sequence set forth in SEQ ID NO:2, it is identified whether the nucleotide represented by Nucleotide Number 63008 is G or T, and it is determined, when the nucleotide is T, that the test subject has a predisposition to curly hair, or when the nucleotide is G, the test subject does not have a predisposition to curly hair;
(11) in the base sequence set forth in SEQ ID NO:3, it is identified whether the nucleotide represented by Nucleotide Number 24524 is T or G, and it is determined, when the nucleotide is G, that the test subject has a predisposition to curly hair, or when the nucleotide is T, the test subject does not have a predisposition to curly hair;
(12) in the base sequence set forth in SEQ ID NO:3, it is identified whether the nucleotide represented by Nucleotide Number 60701 is G or A, and it is determined, when the nucleotide is A, that the test subject has a predisposition to curly hair, or when the nucleotide is G, the test subject does not have a predisposition to curly hair.
In addition, the SNP detected in the method of the present invention for determining the genetic susceptibility (genetic predisposition) to hair shape may be any one of the SNPs described above, or may be two or more thereof. Preferably, two or more SNPs are detected, and thereby, the type or the presence or absence of the genetic predisposition of the test subject to the hair shape, which is a general polygenic trait, can be made clear, while a gene which serves as a main factor determining the hair shape of the test subject can be retrieved with higher accuracy.
The detection of the SNPs can be carried out by directly determining the base sequence of the 1q32.1 to 1q32.2 region of human chromosome 1 further isolated from a sample containing the genomic DNA, or the base sequence of the haplotype block represented by the base sequences set forth in SEQ ID NO:1 to NO:3 in the genomic regions of human chromosome 1. Alternatively, as a method for detecting a polymorphism, in addition to the method of directly determining the gene sequence of the region as described above, there are available a method of determining, when the polymorphism sequence is a restriction enzyme recognition site, the genotype by using the difference in the restriction enzyme cleavage pattern (hereinafter, called RFLP); and methods based on hybridization using a polymorphism-specific probe (for example, a method of determining the type of polymorphism by attaching particular probes on a chip, a glass slide or a nylon film and detecting the difference in the intensity of hybridization with respect to those probes, or a method of determining the genotype by detecting the efficiency of hybridization of a specific probe as the amount of the probe decomposed by a polymerase during amplification of the two strands of a template; a method of detecting the temperature difference in the fusion of two strands by tracing the temperature change of fluorescence emitted by a certain type of two-stranded specific fluorescent dye, and thereby determining the polymorphism; a method of attaching complementary sequences to the two ends of a polymorphic site-specific oligo-probe, and determining the genotype by utilizing the difference between the case where the probe makes a secondary structure within the molecules of the probe itself due to temperature, and the case where the probe hybridizes with the target region; and the like). Further examples include methods of carrying out a nucleotide extension reaction by a polymerase from a template-specific primer, and determining a nucleotide that is accepted to the polymorphic site at that time (a method of using dideoxynucleotides, including fluorescently labeling each of them and detecting the fluorescence of each, and a method of detecting the accepted dideoxynucleotides by mass spectrometry); a method of recognizing the presence or absence of a complementary base pair or a non-complementary base pair at a mutation site by means of an enzyme, subsequent to a template-specific primer, and the like.
Now, conventionally well-known, representative methods for detecting genetic polymorphisms will be listed below, but the present invention is not at all intended to be limited to these: (a) a RFLP (restriction enzyme-cleaved fragment length polymorphism) method; (b) a PCR-SSCP method (analysis of single-stranded DNA higher structure polymorphism, Biotechniques, 16, p. 296-297, 1994, and Biotechniques, 21, p. 510 to 514, 1996); (c) an ASO hybridization method (Clin. Chim. Acta., 189, p. 153-157, 1990); (d) a direct sequencing method (Biotechniques, 11, p. 246-249, 1991); (e) an ARMS method (Nuc. Acids Res., 19, p. 3561-3567, 1991, and Nuc. Acids Res., 20, p. 4831-4837, 1992); (f) a denaturant concentration gradient gel electrophoresis (DGGE) method (Biotechniques, 27, p. 1016-1018, 1999); (g) an RNaseA cleavage method (DNA Cell Biol., 14, p. 87-94, 1995); (h) a chemical cleavage method (Biotechniques, 21, p. 216-218, 1996); (i) a DOL method (Genome Res., 8, p. 549-556, 1998); (j) a TaqMan-PCR method (Genet. Anal., 14, p. 143-149, 1999, and J. Clin. Microbiol., 34, p. 2933-2936, 1996); (k) an invader method (Science, 5109, p. 778-783, 1993, J. Bio. Chem., 30, p. 21387-21394, 1999, and Nat. Biotechnol., 17, p. 292-296, 1999); (l) a MALDI-TOF/MS method (Genome Res., 7, p. 378-388, 1997, and Eur. J. Clin. Chem. Clin. Biochem., 35, p. 545-548, 1997); (m) a TDI method (Proc. Natl. Acad. Sci. USA, 94, p. 10756-10761, 1997); (n) a molecular beacon method (Nat. Biotechnol., 16, p. 49-53, 1998); (O) a dynamic allele specific hybridization (DASH) method (Nat. Biotechnol., 17, p. 87-88, 1999); (p) a padlock probe method (Nat. Genet., 3, p. 225-232, 1998); (q) a DNA chip or DNA microarray (Nakamura, Yusuke, et al., “SNP Idenshi Takei no Senryaku (Strategy for SNP Gene Polymorphism)”, Nakayama Shoten Co., Ltd., p. 128-135, 2000); and (r) an ECA method (Anal. Chem., 72, p. 1334-1341, 2000).
Those described above are representative methods for gene polymorphism detection; however, the method of the present invention for determining the genetic susceptibility (genetic predisposition) to hair shape is not limited to these, and any other gene polymorphism detection methods that are already known or will be developed in the future can be broadly used. Furthermore, in regard to the gene polymorphism detection of the present invention, these methods for gene polymorphism detection may be used singly, or two or more methods can also be used in combination. Hereinafter, as representative methods, the TaqMan-PCR method and the invader method that are used in the Examples described below will be explained in more detail.
(1) TaqMan-PCR Method
The TaqMan-PCR method is a method of using a fluorescent-labeled, allele-specific oligonucleotide (TaqMan probe), and PCR by a Taq DNA polymerase. As the TaqMan probe, an oligonucleotide containing a contiguous base sequence of about 15 to about 30 nucleotides, which is a partial base sequence of a haplotype block represented by any one of SEQ ID NO:1 to NO:3 in the genomic region of human chromosome 1, and contains several polymorphic sites described above (for example, a nucleic acid probe contained in the reagent for hair shape determination of the present invention that will be described below), is used. The probe is labeled with a fluorescent dye such as FAM or VIC at the 5′-terminal, and with a quencher (quenching substance) such as TAMRA at the 3′-terminal, respectively, and in the state as received, since the quencher absorbs the fluorescent energy, fluorescence is not detected. It is preferable to produce probes for both alleles, and to label the probes with fluorescent dyes having different fluorescence wavelengths for batch detection (for example, FAM for one allele and VIC for the other). Furthermore, the 3′-terminal is phosphorylated so that a PCR extension reaction from the TaqMan probe does not occur. When a PCR is carried out using a primer which is designed to amplify a partial sequence of the genomic DNA containing a region that hybridizes with the TaqMan probe, as well as a TaqDNA polymerase, the TaqMan probe hybridizes with the template DNA, and at the same time, an extension reaction from the PCR primer occurs. However, when the extension reaction proceeds, the hybridized TaqMan probe is cleaved due to the 5′ nuclease activation of the Taq DNA polymerase, and the fluorescent dye is released and is no longer affected by the quencher, so that fluorescence is detected. With the amplification of the template, the fluorescence intensity increases exponentially. For example, in the detection of a polymorphism in the nucleotide represented by Nucleotide Number 1 (rs576697, T or C) in the base sequence set forth in SEQ ID NO:1, when an allele-specific oligonucleotide containing the nucleotide (having a length of about 15 to about 30-mers; the C allele is labeled with FAM, and the T allele is labeled with VIC, respectively, at the 5′-terminals, and the 3′-terminals are both labeled with TAMRA) is used as the TaqMan probe, if the genotype of the test subject is CC or TT, high fluorescence intensity of FAM or VIC is recognized in the respective cases, while the other fluorescence is almost unrecognizable. On the other hand, if the genotype of the test subject is CT, fluorescence of both FAM and VIC is detected.
(2) Invader Method
In the invader method, unlike the TaqMan-PCR method, the allele-specific oligonucleotide (allele probe) itself is not labeled, and the oligonucleotide has a sequence having no complementarity to the template DNA on the 5′ side of the nucleotides at the polymorphic site (flap) and has a complementary sequence specific to the template on the 3′ side. In the invader method, use is made of an oligonucleotide having a complementary sequence specific to the 3′ side of the polymorphic site of the template (invader probe; the nucleotides corresponding to the polymorphic site, which is the 5′-terminal of the probe, are arbitrary), and a FRET (Fluorescence Resonance Energy Transfer) probe characterized in that the 5′ side has a sequence capable of adopting a hairpin structure, and the sequence contiguous from the nucleotides forming pairs with the nucleotides of the 5′-terminal to the 3′ side when a hairpin structure is formed, is a sequence complementary to the flap of the allele probe. The 5′-terminal of the FRET probe is fluorescent labeled (for example, FAM, VIC, or the like), and a quencher (for example, TAMRA, or the like) is bonded in the vicinity thereof, so that in the state as received (hairpin structure), fluorescence is not detected. When the template genomic DNA is allowed to react with the allele probe and the invader probe, upon the complementary binding of the three entities, the 3′-terminal of the invader probe penetrates into the polymorphic site. When the single-stranded portion of the allele probe (that is, the flap portion on the 5′ side from the nucleotides of the polymorphic site) is cut using an enzyme which recognizes the structure of this polymorphic site (Cleavase), the flap complementarily binds with the FRET probe, and the polymorphic site of the flap penetrates into the hairpin structure of the FRET probe. When Cleavase recognizes and cleaves this structure, the fluorescent dye used to label the terminal of the FRET probe is released and is no longer affected by the quencher, and thus fluorescence is detected. An allele probe whose nucleotides of the polymorphic site do not match with the template is not cleaved by Cleavase, since an allele probe which is not cleaved can also hybridize with the FRET probe, fluorescence is similarly detected. However, because the reaction efficiency is different, in the allele probe whose nucleotides of the polymorphic site match the template, the fluorescence intensity is markedly stronger than that of the allele probe which does not match. Usually, it is preferable to have the template DNA amplified by PCR using a primer capable of amplifying the region containing the portions where the allele probe and the invader probe hybridize, before the template DNA is allowed to react with the three kinds of probes and Cleavase.
The hair shape of a person can be freely changed by a permanent treatment, a styling agent treatment, brushing or the like, and also can change in an acquired manner, through changes in aging, metabolism, and the like. For this reason, it is difficult to correctly determine or classify the intrinsic natural hair shape of a person based only on the phenotype. Furthermore, since the hair shape can be considered as a general trait of complicated polygenicity, it can be speculated that for individual persons, the gene which serves as a main causative factor for determining the hair shape among the hair shape susceptibility genes of the present invention described above, may vary in different individuals. Therefore, when the genetic predisposition to hair shape is examined and/or determined, a method for regulating the hair shape appropriate for the individuals can be provided.
Furthermore, according to the method, the susceptibility to an acquired change in the hair shape of a test subject, that is, the risk of hair shape change, can be determined. The risk of hair shape change can be mechanically determined using the polymorphisms described above as the reference (index), without requiring the judgment of a person having expertise such as a doctor. Accordingly, the method of the present invention can also be used as a method for detecting the risk of hair shape change.
Through the method of the present invention for determining the genetic susceptibility (genetic predisposition) of a test subject to hair shape, the type or the presence or absence of the genetic predisposition of the test subject to hair shape, which is a general polygenic trait, can be made clear, and a gene which serves as the main causative factor that determines the hair shape of the test subject can be searched among the hair shape susceptibility genes of the present invention. Furthermore, appropriate measures for promoting the regulation of hair shape in the test subject can be devised based on the results of the search. Therefore, the present invention is extremely useful as a method for the examination and/or determination for the fundamental regulation of hair shape.
5. REAGENT FOR DETERMINATION OF GENETIC SUSCEPTIBILITY (GENETIC PREDISPOSITION) TO HAIR SHAPE AND KIT INCLUDING THE REAGENT
The present invention also provides a reagent to be used in the determination method of the present invention, and a kit including the reagent. That is, the reagent for determination of the invention and the kit including the reagent include a nucleic acid probe and/or a primer capable of detecting one or more SNPs selected from the group consisting of an SNP in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) of human chromosome 1, which is determined by a linkage disequilibrium analysis on a single polynucleotide polymorphism (SNP) marker whose allele frequency is statistically significantly different between a group having a curly hair trait and a group having a non-curly hair trait, and is present in a haplotype block having a 3,926-bp base sequence set forth in SEQ ID NO: 1, a 76,945-bp base sequence set forth in SEQ ID NO:2, or a 68,637-bp base sequence set forth in SEQ ID NO:3, and which has a higher allele frequency in an arbitrary curly hair people group than in an arbitrary non-curly hair people group, and an SNP linked to the SNP.
According to an embodiment, the nucleic acid probe used in the reagent for determination of the present invention and the kit including the reagent, is a nucleic acid which specifically hybridizes with the region of a genomic DNA containing the nucleotides of the SNP site to be detected in the method for examination and/or determination of the present invention, and is, for example, a probe which specifically hybridizes with the hair shape determining marker sequence of the present invention. The nucleic acid probe is not particularly limited in the length (length of nucleotides in the portion that hybridizes with the genomic DNA), as long as the nucleic acid probe is specific to a target site to be hybridized and can easily detect polymorphisms. For example, the length is about 10 nucleotides or more, preferably about 15 nucleotides or more, more preferably about 15 to about 600 nucleotides, even more preferably about 15 to about 200 nucleotides, and still more preferably about 15 to about 50 nucleotides. Meanwhile, the phrase “specifically hybridizes with a target site (sequence)” means that cross-hybridization with another DNA does not occur significantly under standard hybridization conditions, preferably under stringent hybridization conditions (for example, conditions described in Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual (3 Vol. set), Cold Spring Harbor Laboratory, NY, 2001). Suitably, the nucleic acid probe preferably has a base sequence complementary to the base sequence of a region containing nucleotides of the polymorphic site to be detected; however, if such specific hybridization is possible, the nucleic acid probe does not need to be completely complementary.
The nucleic acid probe may contain an additional sequence appropriate for the detection of polymorphism (a sequence which is not complementary to the genomic DNA). For example, the allele probe used in the invader method has an additional sequence called flap, at the 5′-terminal of the nucleotides of the polymorphic site. Furthermore, the probe may also be labeled with an appropriate labeling agent, for example, a radioisotope (for example, 125I, 131I, 3H, and 14C), an enzyme (for example, β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase, malate dehydrogenase, or the like), a fluorescent substance (for example, fluorescamine, fluorescein isothiocyanate, or the like), or a luminescent substance (for example, luminol, a luminol derivative, luciferin, lucigenin, or the like). Alternatively, the probe may also be further bonded, in the vicinity of a fluorescent substance (for example, FAM, VIC, or the like), with a quencher (quenching substance) which absorbs the fluorescent energy emitted by the fluorescent substance. In such an embodiment, the fluorescent substance and the quencher are separated at the time of the detection reaction, and fluorescence is detected.
The nucleic acid probe can also be used after being immobilized on an arbitrary solid phase. For this reason, the reagent of the present invention and the kit including the reagent can be provided as an immobilized probe in which the probe is immobilized on an arbitrary solid support (for example, a gene chip, a cDNA microarray, an oligo-DNA array, a membrane filter, or the like, on which a probe is immobilized). Suitably, the immobilized probe is provided as a DNA chip for hair shape susceptibility gene detection.
The solid support used in immobilization is not particularly limited as long as nucleic acid can be immobilized thereon, and examples include a glass plate, a nylon membrane, microbeads, a silicon chip, a capillary, other supports, or the like. The immobilization of a nucleic acid on a solid support may be carried out by a method of mounting a previously synthesized nucleic acid on a solid phase, or by a method of synthesizing a target nucleic acid on a solid phase. The immobilization method is, for example, in the case of a DNA microarray, well known in the art according to the type of the immobilization probe, e.g., a commercially available spotter (manufactured by Amersham Biosciences Corp.), or the like (for example, in situ synthesis of oligonucleotides by photolithographic technology (Affymetrix, Inc.) or inkjet technology (Rosetta Inpharmatics, Inc.), and the like).
The nucleic acid primer used in the reagent for determination of the present invention and the kit including the reagent, may be any nucleic acid primer as long as it is designed to be capable of specifically hybridizing with the region of a genomic DNA containing the nucleotides of the SNP site to be detected in the method for examination and/or determination of the present invention, and specifically amplifying the nucleic acid sequence. For example, the primer is a primer which specifically hybridizes with the nucleic acid sequence of the hair shape determining marker of the present invention and amplifies the hair shape determining marker. Here, the phrase “specifically hybridizes with a target site (sequence)” means that cross-hybridization with another DNA does not occur significantly under the standard hybridization conditions, preferably under stringent hybridization conditions (for example, the conditions described in Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual (3 Vol. set), Cold Spring Harbor Laboratory, NY, 2001).
The method for amplifying the nucleic acid sequence using a primer is not particularly limited as long as it is a method ordinarily used in the art. For example, generally, a PCR method is broadly used, but examples include RCA (Rolling Circle Amplification; Proc. Natl. Acad. Sci., Vol. 92, 4641-4645 (1995)), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), LAMP (Loop-Mediated Isothermal Amplification of DNA; Bio Industry, vol. 18, No. 2 (2001)), NASBA (Nucleic acid Sequence-based Amplification method; Nature, 350, 91-(19.91)), TMA (Transcription Mediated Amplification method; J. Clin. Microbiol. Vol. 31, 3270-(1993), and the like). The number and type of the nucleic acid primer required for amplification can vary depending on the amplification method. For example, in the case of using a PCR method, the required primer may be a pair of nucleic acid primers, which is a combination of a nucleic acid containing a base sequence having about 10 to about 50 nucleotides, preferably about 15 to about 50 nucleotides, and more preferably about 15 to about 30 nucleotides, that is a partial base sequence of a haplotype block represented by a base sequence set forth in any one of SEQ ID NO:1 to NO:3 in the genomic region of human chromosome 1, and specifically hybridizes with a portion of the complementary strand sequence on the 5′ side relative to the nucleotides of the polymorphic site to be detected, and a nucleic acid containing a base sequence having about 10 to about 50 nucleotides, preferably about 15 to about 50 nucleotides, and more preferably about 15 to about 30 nucleotides, that is the partial base sequence and specifically hybridizes with a portion of the complementary strand sequence on the 3′ side relative to the nucleotides of the polymorphic site, the fragment of the nucleic acid to be amplified by the combination of nucleic acids having a length of about 50 to about 1000 nucleotides, preferably about 50 to about 500 nucleotides, and more preferably about 50 to about 200 nucleotides.
The primer may also contain an additional sequence appropriate for the detection of polymorphism (a sequence that is not complementary to the genomic DNA), for example, a linker sequence. Furthermore, the primer may also be labeled with an appropriate labeling agent, for example, a radioisotope (for example, 125I, 131I, 3H, or 14C), an enzyme (for example, β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase, or malate dehydrogenase), a fluorescent substance (for example, fluorescamine, or fluorescein isothiocyanate), or a luminescent substance (for example, luminol, a luminol derivative, luciferin, or lucigenin).
Preferably, the nucleic acid probe and/or primer used in the reagent for determination of the present invention and the kit including the reagent include the hair shape susceptibility SNP marker of the present invention, that is, the nucleotides shown below:
(1) in the base sequence set forth in SEQ ID NO:1, nucleotides represented by Nucleotide Numbers 1 (dbSNP Database ID:rs576697, T or C), 1635 (rs645390, G or A), 2527 (rs3767542, G or A), and 3766 (rs675508, C or A);
(2) in the base sequence set forth in SEQ ID NO:2, nucleotides represented by Nucleotide Numbers 7519 (rs2271763, G or A), 16901 (rs10920260, T or G), 30270 (rs16849387, A or G), 31333 (rs12127375, C or G), 50038 (rs1495840, T or A), and 63008 (rs10920269, G or T); and
(3) in the base sequence set forth in SEQ ID NO:3, nucleotides represented by Nucleotide Numbers 24524 (rs3805, T or G), and 60701 (rs823114, G or A).
More preferably, the nucleic acid probe and/or primer used in the reagent for determination of the invention and the kit including the reagent, contains a nucleotide represented by Nucleotide Number 1 (rs576697, T or C) in the base sequence set forth in SEQ ID NO:1; a nucleotide represented by Nucleotide Number 50038 (rs1495840, T or A) in the base sequence set forth in SEQ ID NO:2; and a nucleotide represented by Nucleotide Number 60701 (rs823114, G or A) in the base sequence set forth in SEQ ID NO:3.
As the nucleic acid probe having the nucleotides of the polymorphic sites described above, a nucleic acid having the nucleotides of any one of the alleles for various polymorphic sites can be used, or two nucleic acids having the nucleotides each respectively corresponding to each of the alleles can also be used, depending on the method for detecting polymorphism used. Meanwhile, in regard to the invader probe used in the invader method, the nucleotides of the polymorphic site (that is, the nucleotides at the 3′-terminal) may be any arbitrary nucleotides.
The nucleic acid probe and/or primer used in the reagent for determination of the present invention and the kit including the reagent may be a DNA or an RNA, and may be single-stranded or double-stranded. In the case of being double-stranded, the nucleic acid probe and/or primer may be any one of a double-stranded DNA, a double-stranded RNA, and a DNA/RNA hybrid. The nucleic acid probe and/or primer can be produced, based on the information of the base sequence, according to an ordinary method using, for example, a commercially available nucleotide synthesizer.
The nucleic acid probe and/or primer described above can be respectively separately (or if possible, in a mixed state) dissolved in water or an appropriate buffer solution (for example, TE buffer, or the like) to an appropriate concentration (for example, 1 to 50 μM, or the like at 2 to 20× concentration), and can be stored at about −20° C. The reagent for determination of the present invention and the kit including the reagent may further include, as constituents, other components necessary for carrying out the method, for example, a buffer for hybridization reaction, an enzyme for nucleic acid amplification reaction, a buffer and other necessary reagents, a reagent for labeling, a reagent for label detection, and apparatuses needed for those reactions or procedure, depending on the method for detecting polymorphism used. For example, when the reagent and the kit including the reagent are for polymorphism detection according to a TaqMan-PCR method, the reagent and the kit including the reagent can further include a 10×PCR reaction buffer solution, a 10× aqueous solution of MgCl2, a 10× aqueous solution of dNTPs, a Taq DNA polymerase (5 U/μL) and the like.
The reagent for determination of the present invention and the kit including the reagent can be used for the examination and/or determination of the genetic susceptibility (genetic predisposition) to hair shape.
6. USE OF HAIR SHAPE SUSCEPTIBILITY GENE OR PROTEIN ENCODING THE GENE
In regard to the hair shape susceptibility gene identified by the procedure described above or an expression product thereof, the expression or activity changes in association with the hair shape. Therefore, the hair shape susceptibility gene and an expression product thereof can be used as a marker for the type of hair shape for detecting and/or determining the type of hair shape of a test subject. Alternatively, when the amount of expression of the hair shape susceptibility gene or an expression product thereof is measured and evaluated, the evaluation or selection of a regulating agent for the hair shape of a person can be carried out. Furthermore, alternatively, when the amount of expression of the hair shape susceptibility gene or an expression product thereof is controlled, the hair shape of a person can be regulated.
According to the present invention, the person who can serve as an object in need of the detection and/or determination of the type of hair shape or the regulation of hair shape, is not particularly limited to a specific human race or group, but Asian race is preferred, while Japanese people are more preferred.
The hair shape susceptibility gene and an expression product thereof that are used as the hair shape determining marker may be a gene which overlaps with the haplotype block having a base sequence set forth in any one of SEQ ID NO:1 to NO:3 or an expression product thereof. However, preferred examples include CSRP1 gene, NAV1 gene, IPO9 gene, TMEM58 gene and NUCKS1 gene, and expression products thereof, and among these, CSRP1 gene, IPO9 gene and NUCKS1 gene, and expression products thereof, are more preferred.
CSRP1 gene is a gene containing a polynucleotide set forth in SEQ ID NO:42, and CSRP1 protein encoded by the gene has an amino acid sequence set forth in SEQ ID NO:43. CSRP1 gene is reported as a gene which has a LIM domain that is believed to function in various scenes including the transcription or generation of genes, through protein-protein recognition or cytoskeleton interaction, and for which a possibility of participation in the regulation processes important for the generation and cellular differentiation is suggested (Wang X. et al., J. Biol. Chem., 267(13), p. 9176-84, 1992). The gene can be accessed at the NCBI gene database under GeneID: 1465. The gene can be acquired by a known technique for gene manipulation. CSRP1 protein can be obtained by expressing a gene containing a polynucleotide set forth in SEQ ID NO:42, or can also be produced by a general chemical synthesis method, according to the amino acid sequence information set forth in SEQ ID NO:43.
As shown in the Examples that will be described below, gene expression in the hair root areas of Japanese curly hair people and Japanese non-curly hair people was analyzed, and it was found that as compared with the non-curly hair group, the amount of expression of CSRP1 gene is significantly higher in the curly hair group. Further, when a substance having a hair straightening action, such as Amomum cardmomum, is administered, curly hair is alleviated, and the amount of expression of CSRP1 gene is decreased.
IPO9 gene is a gene containing a polynucleotide set forth in SEQ ID NO:44, and IPO9 protein encoded by the gene has an amino acid sequence set forth in SEQ ID NO:45. IPO9 gene is reported to have a function that is responsible for material transfer from the cytoplasm to the inside of the nucleus (Okada N. et al., J. Cell. Mol. Med. 12(53), p. 1863-71, 2008). The gene can be accessed at the NCBI gene database under GeneID: 55705. The gene can be acquired by a known technique for gene manipulation. IPO9 protein can be obtained by expressing a gene containing a polynucleotide set forth in SEQ ID NO:44, or can also be produced by a general chemical synthesis method according to the amino acid sequence set forth in SEQ ID NO:45.
As shown in the Examples that will be described below, gene expression in the hair root area of Japanese curly hair people and Japanese non-curly hair people was analyzed, and it was found that as compared with the non-curly hair group, the amount of expression of IPO9 gene is significantly lower in the curly hair group. Further, when a substance having a hair straightening action, such as Centipeda minima, is administered, curly hair is alleviated, and the amount of expression of IPO9 gene is increased.
NUCKS1 gene is a gene containing a polynucleotide set forth in SEQ ID NO:46, and NUCKS1 protein encoded by the gene has an amino acid sequence set forth in SEQ ID NO:47. NUCKS1 gene is reported as a gene that encodes a highly phosphorylated DNA-binding protein present in the nucleus (Ostvold A C et al., Eur. J. Biochem. 268 (8), p. 2430-40, 2001). The gene can be accessed at the NCBI gene database under GeneID: 64710. The gene can be acquired by a known technique for gene manipulation. NUCKS1 protein can be obtained by expressing a gene containing a polynucleotide set forth in SEQ ID NO:46, or can also be produced by a general chemical synthesis method according to the amino acid sequence set forth in SEQ ID NO:47.
As shown in the Examples that will be described below, gene expression in the hair root area of Japanese curly hair people and Japanese non-curly hair people was analyzed, and it was found that as compared with the non-curly, hair group, the amount of expression of NUCKS1 gene is significantly lower in the curly hair group. Further, when a substance having a hair straightening action, such as Amomum cardamorzium, is administered, curly hair is alleviated, and the amount of expression of NUCKS1 gene is increased.
(1) Polynucleotide Marker for Detecting and/or Determining Type of Hair Shape
According to the present invention, the marker for detecting and/or determining the type of hair shape (marker for the type of hair shape) may be a polynucleotide having the base sequence of the hair shape susceptibility gene of the present invention, or a partial polynucleotide thereof. For example, examples of the marker for the type of hair shape of the invention include a polynucleotide consisting of the base sequences of CSRP1 gene, NAV1 gene, IPO9 gene, TMEM58 gene or NUCKS1 gene; preferably a polynucleotide consisting of the base sequences of CSRP1 gene, IPO9 gene or NUCKS1 gene; and more preferably a polynucleotide consisting of the base sequences set forth in SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, polynucleotides consisting of base sequences complementary to these, and partial polynucleotides thereof.
Furthermore, the marker for the type of hair shape of the present invention can contain a strain consisting of a base sequence which is in a further complementary relation with respect to the base sequence of the polynucleotide consisting of complementary base sequence or a partial polynucleotide thereof described above.
The polynucleotides described above and complementary strands thereof may be respectively used as the marker of the present invention in a single-stranded form, or may also be used as the marker of the present invention in a double-stranded form.
Examples of the partial polynucleotide include a partial polynucleotide of the polynucleotide consisting of the base sequence of the hair shape susceptibility gene of the present invention or a base sequence complementary to this, in which the partial polynucleotide has, for example, a length of contiguous 15 nucleotides or more. The length of the partial polynucleotide can be appropriately set in accordance with the use.
(2) Primer for Amplifying Marker for Type of Hair Shape, and Probe for Detecting the Marker
A partial polynucleotide of the polynucleotide consisting of the base sequence of the hair shape susceptibility gene of the present invention or a base sequence complementary to this, can serve as a primer for amplifying the marker for the type of hair shape. Preferably, the primer amplifies a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a base sequence complementary to this, or a partial polynucleotide of such a polynucleotide.
Furthermore, a polynucleotide consisting of the base sequence of the hair shape susceptibility gene of the present invention or a base sequence complementary to this, or a partial polynucleotide thereof, can serve as a probe for detecting the marker for the type of hair shape. Preferably, the probe detects a polynucleotide consisting of a base sequence set forth in SEQ ID NO: 42, SEQ ID NO:44, or SEQ ID NO:46, or a base sequence complementary to this, or a partial polynucleotide of such a polynucleotide.
That is, a primer for specifically recognizing and amplifying an RNA produced as a result of the expression of CSRP1 gene, IPO9 gene or NUCKS1 gene, or a polynucleotide derived therefrom, or a probe for specifically detecting the RNA or the polynucleotide derived therefrom, is included the primer or probe described above.
Specifically, the polynucleotide or partial polynucleotide can be used as a primer or a probe according to a standard method, in the methods known to specifically detect a particular gene, such as a Northern Blotting method, an RT-PCR method, and an in situ hybridization method.
In the case of using the polynucleotide or partial polynucleotide as a primer, the nucleotide length thereof is usually 15 to 100 nucleotides, preferably 15 to 50 nucleotides, and more preferably 15 to 35 nucleotides.
Furthermore, in the case of using the polynucleotide or partial polynucleotide as a detection probe, one having a nucleotide length of usually 15 nucleotides or more, preferably 15 to 1000 nucleotides, and more preferably 100 to 1000 nucleotides, may be used.
Here, the term “specifically recognizes” means that, as in the case where, for example, in a Northern Blotting method, a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or a base sequence complementary to this, or a partial polynucleotide thereof can be specifically detected, and as in the case where, for example, in an RT-PCR method, the polynucleotide is specifically produced, the detected substance or the product can be considered as a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or a base sequence complementary to this, or a partial polynucleotide thereof.
The partial polynucleotide of a polynucleotide consisting of a base sequence set forth in SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or a base sequence complementary to this, can be designed based on the base sequence of CSRP1 gene, IPO9 gene or NUCKS1 gene as set forth in the sequence numbers described above, for example, through the software program of Primer 3 or Vector NTI. The candidate sequence of the primer or probe thus obtainable, or a sequence containing the sequence in a portion, can be designed as a primer or a probe.
(3) Polypeptide Marker for Detecting and/or Determining Type of Hair Shape
Like the hair shape susceptibility genes listed above, expression products of these genes (proteins encoded by the hair shape susceptibility genes, or polypeptides derived therefrom, or partial polypeptides thereof) can also serve as the marker (polypeptide) for the type of hair shape.
Examples of the expression products include CSRP1 protein, NAV1 protein, IPO9 protein, TMEM58 protein, and NUCKS1 protein (or also referred to as CSRP1, NAV1, IPO9, TMEM58 and NUCKS1), which are proteins encoded by CSRP1 gene, NAV1 gene, IPO9 gene, TMEM58 gene or NUCKS1 gene, respectively; polypeptides derived from these proteins; and partial polypeptides thereof. Preferred examples include CSRP1, IPO9 and NUCKS1, polypeptides derived from these, and partial polypeptides thereof.
More preferably, the expression products are proteins encoded by polynucleotides consisting of base sequences set forth in SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:46, and even more preferably, proteins consisting of amino acid sequences set forth in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47.
Furthermore, the expression products also include proteins which have amino acid sequences resulting from deletions, substitutions or additions of one or several amino acids in the amino acid sequences set forth in SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, and having biological functions equivalent to and/or having equivalent immunological activity to those of proteins consisting of the amino acid sequences set forth in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47 (so-called homologues of CSRP1, IPO9 or NUCKS1).
Here, examples of proteins which have equivalent biological functions include proteins that are equivalent to CSRP1, IPO9 or NUCKS1 in terms of the biochemical or pharmacological functions. Further, examples of proteins having equivalent immunological activity include proteins that have an ability to induce a specific immune reaction in an appropriate animal or cells thereof, and to bind specifically to the antibodies to CSRP1, IPO9 or NUCKS1.
Meanwhile, an indicator that determines the substitution, insertion or deletion of amino acid residues can be found by using a computer program well known to those having ordinary skill in the art, for example, DNA Star software program. For example, the number of variations is typically 10% or less of the total number of amino acids, preferably 5% or less of the total number of amino acids, and more preferably 1% or less of the total number of amino acids. Furthermore, from the viewpoint of maintaining the structure of protein, the amino acid to be substituted is preferably an amino acid having properties that are similar to those of amino acids before substitution in terms of the polarity, charge, solubility, hydrophobicity, hydrophilicity, amphiphilicity and the like of the amino acid.
The partial polypeptide may be a polypeptide consisting of at least 5 contiguous amino acids, and preferably 10 to 100 amino acids, in an amino acid sequence encoded by the hair shape susceptibility gene of the invention (for example, an amino acid sequence set forth in SEQ ID NO:43, SEQ ID NO: 45 or SEQ ID NO:47), and having a biological function and/or immunological activity equivalent to those of an expression product of the hair shape susceptibility gene of the invention (for example, CSRP1, IPO9 or NUCKS1).
The polypeptide encoded by the hair shape susceptibility gene of the present invention can be obtained by operations of DNA cloning, establishment of various plasmids, transfection of the plasmid to a host, culture of the transformant, and collection of protein from the culture, based on the base sequence information of the hair shape susceptibility gene. These operations can be carried out according to known methods, for example, the methods described in Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983); DNA Cloning, D M. Glover, IRL PRESS (1985); and the like.
Specifically, the polypeptide can be obtained by producing a recombinant DNA (e.g., expression vector) that can be expressed by a gene encoding CSRP1, IPO9 or NUCKS1 in a desired host cell is produced, introducing this into a host cell to thereby transform the recombinant DNA, culturing the transformant, and collecting.
Furthermore, the polypeptide encoded by the hair shape susceptibility gene of the present invention can also be produced by a general chemical synthesis method in accordance with an amino acid sequence encoded by the hair shape susceptibility gene.
(4) Antibody Specifically Recognizing Marker (Polypeptide) for Type of Hair Shape
An antibody which specifically recognizes a polypeptide consisting of an amino acid sequence encoded by the hair shape susceptibility gene of the present invention or a partial polypeptide thereof, may be an antibody for detecting the marker (polypeptide) for the type of hair shape described above.
As will be described below, when such an antibody is used, the presence or absence of the expression of the marker (polypeptide) for the type of hair shape (for example, CSRP1, IPO9, NUCKS1, or a polypeptide derived therefrom, or a partial polypeptide thereof) in a tissue of a test subject, and the level of the expression of the marker can be detected. Specifically, when a portion of the hair root area of a test subject or the like is collected by a biopsy method or the like, a protein is produced therefrom according to an ordinary method, and the antibody of the present invention is used according to an ordinary method in, for example, a known detection method such as a Western Blotting method or an ELISA method, the marker (polypeptide) for the type of hair shape present in the tissue can be detected.
The antibody for the detection of the type of hair shape may be a polyclonal antibody or a monoclonal antibody, which are both directed to the marker (polypeptide) for the type of hair shape as an immunizing antigen.
These antibodies can be produced according to known methods (Current protocols in Molecular Biology, edited by Ausubel et al., (1987) published by John Wiley and Sons, Section 11.12-11.13). Specifically, a polyclonal antibody can be obtained by immunizing a non-human animal such as rabbit with using a polypeptide consisting of an amino acid sequence encoded by the hair shape susceptibility gene of the invention (for example, CSRP1, IPO9 or NUCKS1), which has been expressed in Escherichia coli or the like and purified by ordinary methods, or with synthesizing a partial polypeptide of the polypeptide above synthesized according to an ordinary method, and collecting the polyclonal antibody from the blood serum of the immunized animal according to an ordinary method.
On the other hand, a monoclonal antibody can be obtained from a hybridoma cell prepared by immunizing a non-human animal such as a mouse with the polypeptide expressed in Escherichia coli or the like and purified according to ordinary methods as described above, or a partial polypeptide thereof, and subjecting spleen cells obtained from the animal and myeloma cells to cell fusion (Current protocols in Molecular Biology, edited by Ausubel et al., (1987), published by John Wiley and Sons, Section 11.4-11.11).
The partial polypeptide used herein is an oligopeptide having a partial amino acid sequence of a polypeptide consisting of an amino acid sequence encoded by the hair shape susceptibility gene of the invention (for example, CSRP1, IPO9 or NUCKS1). It is not necessary for the partial polypeptide to have a functional biological activity, but it is preferable that the partial polypeptide have the same immunogenic characteristics as those of proteins consisting of the amino acid sequences described above. For example, there may be mentioned an oligopeptide consisting of at least 8 contiguous amino acids, preferably 15 amino acids, and more preferably 20 amino acids, in the amino acid sequences described above, which oligopeptide has immunogenic characteristics equivalent to those of proteins consisting of the amino acid sequences described above, and preferably CSRP1, IPO9 or NUCKS1.
The production of an antibody to such a partial polypeptide can be carried out by increasing the immunological response using various adjuvants depending on the host. Although there are no limitations, examples of such adjuvants include Freund's adjuvant; mineral gels such as aluminum hydroxide; surface-active substances such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsifying agents, keyhole limpet hemocyanin, and dinitrophenol; and human adjuvants such as bacillus Calmette-Guerin (BCG) and corynebacterium parvum. 
(5) Detection and/or Determination of Type of Hair Shape
Detection/determination of the type of hair shape involves collecting a portion of hair root tissue or the like of a test subject by a biopsy method or the like, and detecting and/or determining the type of hair shape by using the marker for the type of hair shape of the present invention contained in the tissue as an indicator. For example, in the method described above, the type of hair shape is detected and/or determined by measuring the expression level (amount of expression) of the hair shape susceptibility gene of the invention (for example, CSRP1 gene, IPO9 gene or NUCKS1 gene), a complementary strand thereof, or a partial polynucleotide thereof, or the amount of expression of a protein derived from the gene (for example, CSRP1, IPO9 or NUCKS1), a homologue thereof, or a partial polypeptide thereof.
Furthermore, the method for detection/determination of the present invention is also used, for example, in the case where a pharmaceutical product, a cosmetic product or the like for alleviating curly hair is administered to a curly hair person, so as to determine the presence or absence or the degree of an alleviation of the curly hair.
1) Biological Sample
Examples of the biological sample used herein include epithelial tissue or epithelial cells of a test subject, for example, a tissue containing cells that are capable of expressing the hair shape susceptibility gene of the invention (for example CSRP1 gene, IPO9 gene or NUCKS1 gene), such as the hair root area or skin; an RNA produced from this tissue; a polynucleotide further produced from the RNA. These RNA, polynucleotide and protein can be prepared, for example, by collecting a portion of the hair root area of a test subject by a biopsy method or the like, and then according to ordinary methods.
2) Detection and/or Measurement of Marker
The detection and measurement of a marker may vary depending on the type of the biological sample used as the object of measurement, and specifically, the detection and measurement are carried out as follows.
(i) Case of Using RNA as Biological Sample of Measurement
In the case of using an RNA as a biological sample, the detection and measurement is carried out by detecting and measuring the expression level of a marker (polynucleotide) for the type of hair shape of the invention in the RNA, for example, CSRP1 gene, IPO9 gene, NUCKS1 gene, or a partial polynucleotide.
Here, specifically, the measurement of the amount of expression of the market can be carried out by carrying out a known method such as a Northern Blotting method, an RT-PCR method, a DNA chip analysis method, or an in situ hybridization analysis method, using a primer for amplifying a polynucleotide that can serve as the marker of the present invention described above, or a probe for detecting the polynucleotide.
In the case of using a Northern Blotting method, when the probe of the invention is used, the presence or absence of the expression of the marker (for example, CSRP1 gene, IPO9 gene, NUCKS1 gene, or a partial polynucleotide thereof) in the RNA, and the level of the expression can be detected and measured.
Specifically, there may be mentioned a method in which, first, the probe DNA is labeled with a radioisotope (32P, 33P, or the like; RI), a fluorescent substance or the like; subsequently, the labeled disease marker thus obtainable is hybridized with an RNA derived from a biological tissue of a test subject that has been transferred onto a nylon membrane or the like according to an ordinary method; and then the double strand of the labeled disease marker (DNA) and the RNA thus formed is detected and measured by measuring the signal originating from the labeled material (RI, a fluorescent substance or the like) of the labeled disease marker with a radiation detector (BAS-1800 II, manufactured by Fujifilm Holdings Corp.), a fluorescence detector or the like.
Furthermore, a method using an AlkPhos DirectTM Labelling and Detection System (manufactured by Amersham Pharamcia Biotech, Inc.) can also be available, in which the method includes labeling a probe DNA according to the protocol of AlkPhos DirectTM, hybridizing the probe DNA with an RNA derived from a biological tissue of a test subject, and then detecting and measuring the signal originating from the labeled material of the probe DNA with a multibioimager STORM860 (manufactured by Amersham Pharmacia Biotech, Inc.).
In the case of using an RT-PCR method, the presence or absence of the expression of the marker in the RNA, and the level of the expression can be detected and measured using the primer of the present invention. Specifically, first, a cDNA is prepared from an RNA derived from a biological tissue of a test subject according to an ordinary method, and by using this cDNA as a template, a pair of primers (a forward strand which binds to the cDNA (minus strand) and a reverse strand which binds to the plus strand) prepared from the marker polynucleotide of the present invention is hybridized with the cDNA, so that the region of the target marker can be amplified. Thereafter, a PCR method is carried out according to an ordinary method, and thus the amplified double-stranded DNA thus obtained is detected.
For the detection of the amplified double-stranded DNA, a method of detecting a labeled double-stranded DNA produced by carrying out the PCR using primers which have been labeled in advance with RI, a fluorescent substance or the like; a method of transferring the produced double-stranded DNA onto a nylon membrane or the like according to an ordinary method, hybridizing this double-stranded DNA by using a labeled disease marker as a probe, and detecting the hybridization product; and the like can be used. The labeled double-stranded DNA product thus produced can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems, Inc.) or the like. Furthermore, an RT-PCR reaction solution is prepared using SYBR (registered trademark) Green RT-PCR Reagents (manufactured by Applied Biosystems, Inc.) according to the protocol, the reaction liquid is allowed to react with ABI PRIME (registered trademark) 7700 Sequence Detection System (manufactured by Applied Biosystems), and the reaction product may be detected. The detection and measurement of the level of expression of the marker (polynucleotide) for the type of hair shape of the present invention in the RNA of a test subject using such an RT-PCR method, will be described in Examples.
In the case of using a DNA chip analysis, a DNA chip bonded with the DNA probe (single-stranded or double-stranded) of the present invention is provided, and this is hybridized with a cRNA prepared from an RNA derived from a biological tissue of a test subject according to a conventional method, the two strands of the DNA and cRNA thus formed are bound with a labeled probe prepared from the marker polynucleotide of the present invention, and thereby, the presence or absence of the expression of the marker of the present invention and the level of the expression can be detected and measured.
Furthermore, a DNA chip capable of detecting and measuring the level of expression of the marker of the present invention can also be used as the DNA chip. As the DNA chip, for example, GeneChip (registered trademark) Human Genome U133 plus 2 manufactured by Affymetrix, Inc. may be used.
(ii) Case of Using Protein as Biological Sample of Object of Measurement
When a protein is used as an object of measurement, the measurement is carried out by contacting the antibody of the invention with a biological sample, detecting the marker (polypeptide) for the type of hair shape of the invention in the biological sample, which has been bound to the antibody, for example, CSRP1, IPO9, NUCKS1, or a partial polypeptide thereof, and measuring the amount (level) of the marker.
Here, the measurement of the amount of protein binding can be carried out by using a known method such as a Western Blotting method.
The Western Blotting method can be carried out by using the antibody of the present invention as a primary antibody, subsequently; labeling the primary antibody using, as a secondary antibody, an antibody which binds to the primary antibody labeled with a radioisotope such as 125I, a fluorescent substance, an enzyme such as horse radish peroxidase (HRP), or the like; and determining the signals originating from these labeled substances with a radiation meter, a fluorescence detector or the like. Furthermore, after using the antibody of the present invention as the primary antibody, the primary antibody is detected using an ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech, Inc.) according to the protocol, and measurement can be made using a multibioimager STORM 860 (manufactured by Amersham Pharmacia Biotech, Inc.).
3) Determination of Type of Hair Shape
The determination of the type of hair shape can be carried out by comparing the level of the marker of the invention (for example, the level of gene expression of CSRP1 gene, IPO9 gene, or NUCKS1 gene, or the amount of CSRP1, IPO9, or NUCKS1) in a biological sample of a test subject, which has been measured as described above, with the corresponding level of a non-curly hair person, and determining the difference between the two levels.
The comparison of the level of expression of the marker polynucleotide or polypeptide between the biological sample of a test subject and the biological sample of a non-curly hair person can be carried out by carrying out the measurements directed to the biological sample of a test subject and the biological sample of a non-curly hair person in parallel. Furthermore, even if the measurements are not carried out in parallel, the average level or a statistical median value of the level of gene expression of the marker polynucleotide (CSRP1 gene, IPO9 gene, NUCKS1 gene, a partial polynucleotide thereof, or the like) or the level of expression of the marker polypeptide (CSRP1, IPO9, NUCKS1, a partial polypeptide thereof, or the like), which has been determined in advance in the tissues of plural (at least 2, preferably 3 or more, and more preferably 5 or more) non-curly hair persons under the same measurement conditions, can be used for the comparison with the test subjects, as the measured value for the test subject with the level of expression of the marker polynucleotide or polypeptide of a non-curly hair person.
The determination of the type of hair shape of a test subject can be carried out by using, as an index, the extent of increase or decrease (for example, higher or lower by two times or more, and preferably three times or more) in the case of comparing the gene expression level of the marker polynucleotide (CSRP1 gene, IPO9 gene, NUCKS1 gene, a partial polynucleotide thereof, or the like) or the expression level of the marker polypeptide (CSRP1, IPO9, NUCKS1, a partial polypeptide thereof, or the like) in the tissue of the test subject, with the levels of a non-curly hair person.
For example, if the expression level of CSRP1 gene or CSRP1 protein of the test subject is higher than such a level of a non-curly hair person, the test subject can be determined as a curly hair person, or is suspected to have the onset of curly hair in the future.
Furthermore, for example, if the expression level of IPO9 gene or IPO9 protein of the test subject is lower than such a level of a non-curly hair person, the test subject can be determined as a curly hair person, or is suspected to have the onset of curly hair in the future.
Further, for example, if the expression level of NUCKS1 gene or NUCKS1 protein of the test subject is lower than such a level of a non-curly hair person, the test subject can be determined as a curly hair person, or is suspected to have the onset of curly hair in the future.
7. METHOD FOR REGULATING HAIR SHAPE
When the nucleotides located at the hair shape susceptibility SNP marker of the present invention are modified, the hair shape of individuals can be fundamentally regulated.
That is, the present invention also provides a method for regulating the hair shape of an individual. According to an embodiment, the method may be a non-therapeutic method for regulating hair shape for cosmetic purposes, and can be carried out by a beautician or a barber. Meanwhile, according to the present specification, the term “non-therapeutic” is a concept which does not encompass medical acts, that is, acts of remedy to human body through treatment.
The method can be achieved by changing the nucleotides located at the hair shape susceptibility SNP markers of the present invention listed above. The specific technique is not particularly limited as long as it is a method capable of achieving the purpose described above, and conventionally known methods and techniques that will be developed in the future can all be used; however, for example, a method of utilizing genetic recombination may be used.
Alternatively, the method for regulating hair shape of the present invention is carried out by controlling the expression of the hair shape susceptibility gene of the present invention in the hair root area of a person in need of regulation of hair shape (for example, suppression of curly hair or kinky hair, or waving of scalp hair).
For example, in a person who is worried about curly hair or kinky hair, curly hair or kinky hair can be suppressed by inducing or promoting the expression of a hair shape susceptibility gene whose expression contributes to the phenotype of straight hair, for example, IPO9 gene or NUCKS1 gene. Alternatively, curly hair or kinky hair can be suppressed by inhibiting the expression of a hair shape susceptibility gene whose expression contributes to the phenotype of curly hair or kinky hair, for example, CSRP1 gene. On the other hand, in a person who wishes for waving of the scalp hair, waving can be expressed or promoted by inducing or promoting the expression of a hair shape susceptibility gene whose expression contributes to the phenotype of curly hair or kinky hair, for example, CSRP1 gene. Alternatively, waving can be expressed or promoted by inhibiting the expression of a hair shape susceptibility gene whose expression contributes the phenotype of straight hair, for example, IPO9 gene or NUCKS1 gene.
For example, in the case of suppressing curly hair or kinky hair, the expression level of IPO9 gene or NUCKS1 gene in the human hair root area may be brought to a value equal to or higher than the mRNA expression level of the relevant gene in a non-curly hair person, and for example, it is desirable to increase the expression level to a value of about 3 to 10 times higher or more. On the other hand, in the case of intending to promote waving, the expression level of IPO9 gene or NUCKS1 gene may be brought to a value lower than the mRNA expression level of the gene in a non-curly hair person, and for example, it is desirable to decrease the expression level to a value of about 3 to 10 times lower or less.
Furthermore, for example, in the case of suppressing curly hair or kinky hair, the expression level of CSRP1 gene in the human hair root area may be brought to a value equal to or lower than the mRNA expression level of the gene in a non-curly hair person, and for example, it is desirable to decrease the expression level to a value of about 3 to 10 times lower or less. On the other hand, in the case of intending to promote waving, the expression level of CSRP1 gene may be brought to a value higher than the mRNA expression level of the gene in a non-curly hair person, and for example, it is desirable to increase the expression level to a value of about 3 to 10 times higher or more.
The suppression, induction or promotion of the expression of a hair shape susceptibility gene in the human hair root area can be carried out according to an ordinary method. For example, in the suppression of gene, a method based on an antisense nucleotide, for example, a technique based on a method of inhibiting the translation from mRNA, or the like, may be used, and in the induction or promotion, a technique of expressing a hair shape susceptibility gene through gene transduction by means of a viral vector or the like may be used, or the like. Furthermore, in the suppression of the expression of a protein encoded by a hair shape susceptibility gene can be basically realized by a technique of suppressing the expression of the gene, and in the induction or promotion of the expression of the protein, a technique of expressing the gene at a high level, as well as a technique of direct intracutaneous injection of a human recombinant protein of the protein or the like may be used.
The gene transduction utilizing an antisense nucleotide can be carried out in the same manner as in the methods ordinarily used in gene therapy. For example, gene transduction can be carried out by a method of directly administering an antisense oligonucleotide or a chemical modification product thereof into the body of a test subject and thereby suppressing the expression of the hair shape susceptibility gene of the present invention, or a method of introducing an antisense RNA to a target cell of a patient and thereby suppressing the expression of the hair shape susceptibility gene of the present invention in the cell.
Here, the term “antisense nucleotide” encompasses an antisense oligonucleotide, an antisense RNA, an antisense DNA and the like, which all correspond to a portion of at least 8 nucleotides or more in a hair shape susceptibility gene of the present invention. Examples of the chemical modification products thereof include derivatives which are capable of increasing the transferability into cells or stability in the cells, such as phosphorothioates, phosphorodithioates, alkyl phosphotriesters, alkylphosphonates, and alkyl phosphoamidates (“Antisense RNA and DNA”, published by WILEY-LISS, 1992., pp. 1-50; J. Med. Chem. 36, 1923-1937 (1993)).
The antisense nucleotide or a chemical modification product thereof can suppress the expression of a hair shape susceptibility gene, that is, the expression of a protein encoded by a hair shape susceptibility gene, by binding to a sense strand mRNA in a cell, and can thereby control the function (activity) of the protein.
In the method of directly administering an antisense oligonucleotide or a chemical modification product thereof into a living body, an antisense oligonucleotide or a chemical modification product thereof used therein may have a length of preferably 5 to 200 nucleotides, more preferably 8 to 25 nucleotides, and most preferably 12 to 25 nucleotides. Upon the administration, the antisense oligonucleotide or a chemical modification product thereof can be formulated into a preparation using a stabilizer, a buffer solution, a solvent and the like that are ordinarily used.
In the method of introducing an antisense RNA into a target cell of a test subject, the antisense RNA used therein may have a length of preferably 100 nucleotides or more, more preferably 300 nucleotides or more, and even more preferably 500 nucleotides or more. Furthermore, this method encompasses an in vivo method of introducing an antisense gene into the cells of a living body, and an ex vivo method of first introducing an antisense gene into the cells that have been extracted out of body, and returning the cells into the body (see Nikkei Science, April 1994, pp. 20-45; Gekkan Yakuji (Pharmaceuticals Monthly) 36 (1), 23-48 (1994); Jikken Igaku (Experimental Medicine) Special Issue, 12 (15), whole page (1994); and the like). Among these, an in vivo method is preferred, and examples thereof include a viral transduction method (a method of using a recombinant virus) and a non-viral transduction method (see the various documents described above).
As the method of using a recombinant virus, for example, methods of inserting an antisense nucleotide of MLTK gene into the genome of a virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, or Sindbis virus, and introducing the product into the living body, may be used. Among these methods, methods of using retrovirus, adenovirus, adeno-associated virus and the like are particularly preferred. As the non-viral transduction method, a liposome method, a lipofectin method and the like may be used, and particularly, a liposome method is preferred. As other non-viral transduction methods, for example, a microinjection method, a calcium phosphate method, an electroporation method and the like may also be used.
A preparation composition for gene transduction contains, as active ingredients, the antisense nucleotide described above or a chemical modification product thereof, recombinant viruses containing these, infected cells to which these viruses have been introduced, and the like.
The administration of the composition to a test subject can be carried by, for example, intravenous, intraarterial, subcutaneous, or intramuscular administration in an appropriate dosage form such as an injection, and can be introduced by directly administering the composition through the skin of a patient. In the case of employing an in vivo method, the composition for gene transduction can be formulated into a dosage form such as an injection containing an antisense nucleotide of a hair shape susceptibility gene, as well as a form in which, for example, a viral vector containing an antisense nucleotide of a hair shape susceptibility gene that is embedded in a liposome or a membrane-fused liposome (Sendai virus (HVJ)-liposome, or the like). These liposome dosage forms include a suspending agent, a freezing agent, a centrifuge concentration freezing agent, and the like. Furthermore, the composition for gene transduction can also be formulated into a form of a culture fluid of cells infected with a virus to which a vector containing the antisense nucleotide of a hair shape susceptibility gene has been introduced. The amount of administration of the active ingredient in these various preparation forms can be appropriately adjusted on the basis of the severity of the disease intended to treat, the age and body weight of the patient, and the like. Usually, in the case of an antisense nucleotide for a hair shape susceptibility gene, the amount of administration may be an amount by which about 0.0001 to 100 mg, and preferably about 0.001 to 10 mg, is administered once in several days to several months to an adult as a test subject.
In the case of a retrovirus vector containing an antisense nucleotide, the amount can be selected in the range of an amount which gives a retrovirus titer of about 1×103 pfu to 1×1015 pfu per day per kg of the patient's body weight. In the case of a cell having an antisense nucleotide introduced therein, an amount of about 1×104 cells/body to 1×1015 cells/body may be administered.
8. METHOD FOR EVALUATION OR SELECTION OF HAIR SHAPE REGULATING AGENT
The present invention also provides a method for evaluating or selecting a hair shape regulating agent (screening method).
The screening method may be carried out by, for example, steps such as described below:
(a) a step of administering a test substance into a cell containing the hair shape susceptibility gene of the present invention; and
(b) a step of selecting, among the administered test substances, a substance which converts a nucleotide polymorphism of the hair shape susceptibility SNP marker of the present invention present on the hair shape susceptibility gene or the vicinity thereof, for example, on the haplotype block containing the gene, to another polymorphism, as a hair shape regulating agent.
The cell used in the step (a) (step of administering a test substance) may be any cell which can be introduced a haplotype block in the genomic region of human chromosome 1, which is represented by a base sequence set forth in any one of SEQ ID NO:1 to NO:3, or a gene which at least overlaps with the haplotype block, that is, the hair shape susceptibility gene of the present invention, and can retain the gene stably, and there are no particular limitations on the origin of the cell (for example, the cell is not limited to a prokaryotic cell or a eukaryotic cell, or to an insect cell or an animal cell, or the like). Meanwhile, gene transduction, cell culture and the like can be carried out by arbitrarily using any methods conventionally known in the art (for example, Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual (3 Vol. Set), Cold Spring Harbor Laboratory, NY, 2001; The Japanese Tissue Culture Association, Ed., “Technology of Tissue Culture, 3rd Edition, Fundamentals and Applications”, Asakura Shoten, 1996; and the like). The cell can be effectively utilized as a screening tool in the method for evaluating or selecting a substance effective for regulating the hair shape (screening method).
There are no particular limitations on the test substance that is administered. Examples include single compounds such as a natural compound, an organic compound, an inorganic compound, a protein and a peptide; and arbitrary compounds or compositions such as a compound library, expression products of a gene library, a cell extract, a cell culture supernatant, products of a fermentation microorganism, a marine extract, and a vegetable extract.
In regard to the step (b) (step of selecting a hair shape regulating agent), the presence or absence of the conversion of a nucleotide polymorphism and the type of the nucleotide after conversion are detected. The method for detecting the presence or absence of the conversion of a nucleotide polymorphism and the type of the converted nucleotide may be a method of directly measuring the type of nucleotides, or a method capable of indirectly evaluating the change of nucleotides. Examples of the method of directly measuring nucleotides include methods that are well known to those having ordinary skill in the art, such as PCR-SSCP, PCR-RLFP, PCR-SSO, PCR-ASP, a direct sequencing method, SNaPshot, dHPLC, a Sniper method, and a MALDI-TOF/MS method. Examples of the method of indirectly evaluating nucleotides, include methods of measuring a function, activity, the amount of a specific mRNA, or the amount of a protein, which may be produced/increased, or lost/decreased as a result of the conversion of the target nucleotides.
The substance selected by the method can be used as a hair shape regulating agent effective for the regulation of hair shape, and can also be used for the preparation of a pharmaceutical product, a quasi-drug, a cosmetic material, a health food, or the like, which all contain the agent. When the selected substance is further subjected to other pharmacological tests, clinical tests and toxicology tests as necessary, a hair shape regulating agent that is more effective and safe to human beings can be obtained.
Alternatively, the screening method described above can be carried out by using, for example, the expression of a hair shape susceptibility gene of the present invention or a protein encoded by the gene in a tissue or cell capable of expressing the gene or protein, as an indicator.
Specifically, the screening method can be carried out by the following steps (a) to (d):
(a) a step for contacting a test substance with a tissue or cell capable of expressing the hair shape susceptibility gene of the present invention or a protein encoded by the gene;
(b) a step of measuring the amount of expression of the gene or the protein in the tissue or cell;
(c) a step of comparing the amount of expression measured in step (b) with the amount of expression of the gene or the protein in a control tissue or cell which has not been contacted with the test substance; and
(d) a step of selecting, based on the results of step (c), a test substance which decreases or increases the amount of expression of the gene or the protein, as a hair shape regulating agent.
Here, as the tissue or cell capable of expressing the hair shape susceptibility gene of the present or a protein encoded by the gene, the type of the tissue or cell does not matter as long as the tissue or cell which expresses the gene or the protein. However, examples include a tissue or a cell of a mammal, for example, the skin tissue, hair root area tissue (hair follicle tissue), epidermal keratinocytes, hair root area-derived cells, an established epithelial cell line, and the like, all collected from a human being. The cell also includes a transformant which has been transformed with the hair shape susceptibility gene of the present invention (an expression vector having the gene).
The contact between the tissue or cell and a test substance can be carried out by, for example, adding the test substance in advance to a culture fluid to a predetermined concentration, and then placing the tissue or cell in the culture fluid, or by adding the test substance to a culture fluid in which the tissue or cell is placed, to a predetermined concentration.
Examples of the culture fluid include DMEM medium, MCDB medium, Willams' E medium, RPMI1640 medium, DMEM/HamF12 (1:1) medium, various commercially available media for epithelial cells, and the like, and appropriately agar or gelatin may also be added. Furthermore, if necessary, an antibiotic substance, an amino acid, blood serum, a growth factor, a biological extract, and the like may also be added.
Tissue culture can be carried out by, for example, inserting a collected hair root area tissue (hair follicle tissue) into a 24-well plate to which a culture fluid has been added, and culturing the tissue usually for 10 to 30 days, and preferably 1 to 21 days, in a gas phase of air containing CO2 at a temperature of 37° C.
Furthermore, cell culture can be carried out by, for example, inserting cells into a 24-well plate to which a culture fluid has been added, and culturing the cells usually for 1 to 7 days, and preferably 1 to 3 days, in a gas phase of air containing CO2 at a temperature of 37° C.
The measurement (quantification) of the expression of the gene can be carried out according to the method described in connection with the detection/measurement of a marker for the type of hair shape described above ((5)-2)-(i)). That is, the measurement can be carried out by performing a known method such as a Northern Blotting method, an RT-PCR method, a DNA chip analysis method, or an in situ hybridization analysis method, using a primer for amplifying a polynucleotide that can serve as the marker of the present invention, or a probe for detecting the polynucleotide.
Furthermore, the measurement (quantification) of the expression of the protein can be carried out according to the method described in connection with the detection/measurement of a marker for the type of hair shape described above ((5)-2)-(ii)). That is, the measurement can be achieved according to a known method such as a Western Blotting method, using an antibody which recognizes the marker (polypeptide) for the type of hair shape of the present invention.
2) The measurement of the expression level of the hair shape susceptibility gene of the present invention can also be carried out by introducing into a cell line a fusion gene in which a reporter gene such as, for example, luciferase gene, is linked to a gene region controlling the expression of the gene (regulatory region), and measuring the amount or activity of a protein derived from the reporter gene.
That is, the method for evaluating or selecting a hair shape regulating agent according to the present invention can be carried out by the following steps of (a) to (c):
(a) a step of introducing a fusion gene of the regulatory region of a hair shape susceptibility gene of the present invention and a reporter gene, into a cell capable of expressing the hair shape susceptibility gene of the present invention, and culturing the cell in the presence and in the absence of a test substance;
(b) a step of measuring the amount of expression of an expression product of the reporter gene in the cell culture cultured in the presence of the test substance, and comparing the amount with the amount of expression of an expression product of the reporter gene in the cell culture cultured in the absence of the test substance; and
(c) a step of selecting, based on the comparison results obtained in step (b), a test substance which increases or decreases the amount of expression of the reporter gene expression product, as a hair shape regulating agent.
As the reporter gene, a structural gene of an enzyme which catalyzes a light emission reaction or a color reaction is preferred. Specifically, examples include the luciferase gene described above, secreted alkali phosphatase gene, chloramphenichol acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, aequorin gene, and the like.
Furthermore, as the regulatory region of the hair shape susceptibility gene, for example, about 1 kb to about 10 kb, and preferably about 2 kb, upstream of the transcription initiation site of the gene can be used, and for example, the regions having base sequences set forth in SEQ ID NO:48 to NO:50 in CSRP1 gene, IPO9 gene, or NUCKS1 gene, respectively, may be used. The preparation of a fusion gene and the measurement of the activity originating from a reporter gene can be carried out by known methods.
A substance which decreases the amount of expression of the hair shape susceptibility gene may be a substance which suppresses the expression of or promotes the degradation of a mRNA complementary to the polynucleotide constituting the gene, and a substance which decreases the amount of expression of a protein encoded by the hair shape susceptibility gene may be a substance which suppresses the expression of the hair shape susceptibility gene or a protein thereof, or promotes the degradation of the gene or a protein thereof, and consequently decreases the amount of expression of the protein.
A substance which increases the amount of expression of the hair shape susceptibility gene of the present invention may be a substance which promotes the expression of or suppresses the degradation of a mRNA complementary to the polynucleotide constituting the gene, and a substance which increases the amount of expression of a protein encoded by the hair shape susceptibility gene may be a substance which promotes the expression of the hair shape susceptibility gene or a protein thereof, or suppresses the degradation of the gene or a protein thereof, and consequently increases the amount of expression of the protein.
A substance which increases the amount of expression of the hair shape susceptibility gene or a protein encoded by the gene serves as a reducing or promoting agent for curly hair or kinky hair. For example, a substance which increases the amount of expression of CSRP1 gene, IPO9 gene or NUCKS1 gene, or a protein encoded thereby, can serve as a reducing or improving agent for curly hair or kinky hair, while a substance which decreases the expression of such a gene or protein can serve as a promoting agent for curly hair or kinky hair, or a waving promoting agent. Furthermore, for example, a substance which increases the amount of expression of IVL gene or a protein encoded thereby, can serve as a promoting agent for curly hair or kinky hair, or a waving promoting agent, while a substance which decreases the expression of the gene or protein can serve as a reducing or improving agent for curly hair or kinky hair. Such a hair shape regulating agent can function as a pharmaceutical product, a cosmetic product or the like for an amelioration of curly hair or kinky hair, or for the promotion of waving of scalp hair, when administered to a human being.
3) Furthermore, the method for evaluating or selecting the hair shape regulating agent of the present invention can be carried out by using the function (activity) of a protein encoded by the hair shape susceptibility gene of the present invention as an indicator.
Examples of the function or activity of the protein include the acetylcholine receptor activity (Nguyen V T et al., J. Biol. Chem., 275(38), p. 29466-76, 2000), and phosphatidylserine binding ability (Goebeler V et al., FEBS Lett. 546(2-3), p. 359-64, 2003). The amount of the protein and the function or activity therefore have a certain correlation. Therefore, when the measurement of the function or activity of the protein described above is measured instead of the measurement of the amount of the protein, an evaluation or selection of a hair shape regulating agent can be carried out.
Specifically, the evaluation or selection is carried out by the following steps (a), (b) and (c):
(a) a step for contacting a test substance with an aqueous solution, tissue cells, or a cell fraction prepared from the tissue cells containing a protein encoded by the hair shape susceptibility gene of the present invention;
(b) a step of measuring the function or activity of the protein in the aqueous solution, tissue cells or cell fraction that has been contacted with the test substance, and comparing the function or activity with the function or activity of the protein in a control aqueous solution, control cells or control cell fraction which has not been contacted with the test substance; and
(c) a step of selecting, based on the comparison results of the step (b), a test substance which increases or decreases the function or activity of the protein.
As the aqueous solution containing a protein encoded by the hair shape susceptibility gene, examples include aqueous solutions of CSRP1, IPO9, or NUCKS1, as well as a tissue cell lysate, a nucleus extract, and cell culture supernatant, which contain such a protein, and the like. The cell used herein may be a cell which expresses the hair shape susceptibility gene of the invention (for example, CSRP1 gene, IPO9 gene, or NUCKS1 gene), and has a protein encoded by such a gene as an expression product. Specifically, a tissue or cell of a mammal, for example, the skin tissue, hair root area tissue (hair follicle tissue), epidermal keratinocytes, hair root area-derived cells, an established epithelial cell line, and the like, all collected from a human being, can be used. The cell also includes a transformant which has been transformed with the hair shape susceptibility gene of the present invention (or an expression vector having the gene). Examples of host cells used, in the transformation include well known cells such as Hela cell, COS cell, HEK293 cell, MDCK cell, CHO cell, and HL60 cell. Furthermore, a cell fraction means one of various fractions derived from the cells described above, and includes, for example, a cell membrane fraction, a cell cytoplasm fraction, a cell nucleus fraction, and the like.
The activity of a protein encoded by the hair susceptibility gene of the present invention can be measured, for example, in the case of measuring the acetylcholine receptor activity or the phosphatidylserine binding ability, by known methods such as a binding assay, a co-immunoprecipitation method, a pulldown assay, a two-hybrid method (Y2H), a fluorescence polarization method, and a time-resolved fluorescence resonance energy transfer (TR-FRET) method (for example, Hiromitsu Nakauchi, Ed., “Immunological Protocol”, Yodosha Co., Ltd., 2004; Tadaomi Takenawa, Ed., “Optimal Methods Clarifying Protein Interaction”, Biotechnology Journal, Vol. 5, No. 6, Yodosha Co., Ltd., 2005). That is, the activity can be measured by immobilizing a protein encoded by a hair shape susceptibility gene on a membrane or a plate using an aqueous solution containing the protein, and detecting the amount of radioisotope-labeled acetylcholine or phosphatidylserine binding to the protein. A substance which suppresses (decreases) the function (activity) of the protein may be a substance which decreases the acetylcholine receptor activity or the phosphatidylserine binding ability, while a substance which enhances (increases) the function (activity) of the protein may be a substance which increases the acetylcholine receptor activity or the phosphatidylserine binding ability. For example, a substance which enhances the function (activity) of IPO9 or NUCKS1 can serve as an ameliorating agent for curly hair or kinky hair, and a substance which suppresses the function (activity) of such a protein can serve as a waving promoting agent. For example, a substance which enhances the function (activity) of IPO9 or NUCKS1 can serve as an improving agent for curly hair or kinky hair, and a substance which suppresses the function (activity) of such a protein can serve as a waving promoting agent. Furthermore, for example, a substance which enhances the function (activity) of CSRP1 can serve as a waving promoting agent, while a substance which suppresses the function (activity) of such a protein can serve as an ameliorating agent for curly hair or kinky hair.
EXAMPLES
Hereinafter, the present invention will be described by way of Examples.
Example 1
Definition of Hair Shape and Collection of Curly Hair Family Lines
In the present Example, an affected sib-pair linkage analysis and a case-control association analysis were carried out on a Japanese group, in order to identify the hair shape susceptibility gene.
In general, hair shape varies with the human race, and the people of the Asian race relatively more frequently have straight hair, while the people of the African race mainly have kinky hair (or curled hair). A large proportion of the people of the Indo-European race have a trait of wavy hair (wave hair) which is intermediate of the two. Since a Japanese group is a straight hair-dominant group, people having a curly hair trait as the hair shape were defined as the affected (case), while the straight hair trait was defined as the control (control). In a genetic analysis such as a linkage analysis, it is necessary to handle the object traits quantitatively to a certain extent, and thus, for example, a method of binarizing the traits in such a manner that curly hair=1 and straight hair=0, or a method of measuring the degree of curly hair by a certain method, and quantifying the degree were considered. However, in the current situation, various and diverse hair shapes of human being are available, and the method for measurement or classification has not been sufficiently established. Thus, first, an accurate classification of the phenotypes of hair shape was carried out. The hair shape is defined by the overall feature of the hair and the degree of curl (curl radius). Furthermore, factors defining the hair shape include not only the curl characteristics of a single hair, but also the synchrony of curl with the groups of hair in the surroundings. Thus, the phenotypes of hair shape were classified as indicated in Table 4, based on the actual states of hair shape in various human races. This classification is applicable to various racial groups, including Japanese groups. Furthermore, FIG. 1 presents images of the phenotypes of hair shape.







TABLE 4







Classification of phenotypes of hair shape













Type of hair



Feature
Curl radius
shape














Type 1
Hair which exhibits
9.5 cm or
Straight hair



one curl in overall
larger over



even if the length of
the entire



the hair changes, or
hair, or 3 cm



has one curl only at
or larger only



the hair tips
at the hair tip




Smaller than
Almost




9.5 cm over the
straight hair,




entire hair,
or slightly




or smaller
wavy hair




than 3 cm only




at the hair tip


Type 2
Hair which has
9.5 cm or
Almost



several repeated
larger over
straight hair,



curls along the
the entire
or slightly



length of the hair
hair
wavy hair



with an inherent curl
Equal to or
Wavy hair



radius, and has a curl
larger of 3 cm



period synchronizing
and smaller



with the hair in the
than 9.5 cm



surroundings
over the




entire hair




A curl of
Curly hair, or




smaller than 3
strongly wavy




cm in the
hair




entire hair


Type 3
Hair in which

Kinky hair



individual hairs have



finely repeated



curls, and the curl



period does not



synchronize with the



hair in the



surroundings









On the other hand, the phenotype is the hair shape is a quantitative trait which can be continuously changed in a group, and it has been established to which extent should be determined as the curly hair trait or as the straight hair trait. In the present invention, among the classifications based on the actual states of hair shape, kinky hair, and curly hair or strongly wavy hair are defined as the curly hair traits, and wavy hair, almost straight hair or slightly wavy hair, and straight hair are defined as the straight hair (non-curly hair) traits.
As such, the phenotypes of hair shape could be accurately classified, but in regard to the collection of the objects of genetic analysis, the following problem to be solved emerged. That is, problems arise when the hair at the time point of collection is markedly short and it is impossible to evaluate the shape, and when the original hair shape has changed by permanent treatment, hair dyeing, and chemical treatments by various styling agents. For this reason, all candidates who could become the objects of a genetic analysis were each requested to submit a photograph of the candidate himself/herself that was taken at a time when the phenotype of the hair shape could be discriminated (for example, childhood). That is, it is a photograph of a hair state which is not a markedly short hair and has not been subjected to a chemical treatment of hair. At the same time, all of the candidates were requested to submit several hair strands. The submitted hair strands were subjected to a detailed shape evaluation of torsion or kink of the hair, crimp, curl characteristics, and the like under water immersion conditions by which the effect of chemical treatment is lost. The objects of a genetic analysis were determined based on the evaluation of hair shape from the submitted photographs of the candidates themselves, and the evaluation of the shape of the submitted hair, and finally based on an investigation of hair shape through interviews.
As such, it took about two years to collect curly hair family lines of 68 families with 283 members among 3000 or more candidates applied from all over Japan. The specific details include 41 groups of two siblings, 22 groups of three siblings, 4 groups of four siblings, and one group of five siblings, and 100 pairs were defined as the final affected sib-pairs (brothers or sisters having the curly hair trait). Since it was predicted that this number of sib-pairs was sufficient to characterize the genetic locus in consideration of the strength of the genetic factor and the risk in the siblings, it was decided to carry out an affected sib-pair linkage analysis.
In regard to the collection of specimens from the objects of the genetic analysis, specimens were collected only when an approval was granted in advance by the ethics committee, subsequently the person in charge of the implementation of informed consent explained the contents of the study to the objects using a written explanation, and written consent was obtained.
A doctor or a nurse collected about 20 mL of blood from each of the objects of the genetic analysis. The genomic DNA was extracted from the blood specimen using PUREGENE Genomic DNA Purification Kit (manufactured by Gentra Systems, Inc.) according to the manual. The genomic DNA was dissolved in 2 mL of a DNA Hydration Solution, the concentration was measured, and the solution was stored at 4° C. The average yield of the genomic DNA was 576.2 g/20 ml of blood.
Example 2
Affected Sib-Pair Linkage Analysis on Entire Genome
In the present Example, an affected sib-pair linkage analysis covering the entire genome was carried out for the first time on the Japanese curly hair family lines. To briefly describe the principle of this method, since siblings that are affected have inherited from their parents an allele causative of a disease, the siblings necessarily share the allele. On the other hand, the number of alleles shared by brothers is 1 (a value based on the null hypothesis). When many cases of allele sharing could be observed from the number of alleles based on the null hypothesis by examining the number of alleles shared by many affected sib-pairs, it was determined that linkage was recognized.
The affected sib-pair linkage analysis was carried out using a linkage mapping set (ABI PRISM Linkage Mapping Set-MD 10 v2.5) manufactured by Applied Biosystems, Inc. (ABI). This is a set of 400 fluorescent primers for typing in total, intended to amplify microsatellites, which are short repeating sequences rich in polymorphisms that are evenly scattered in the genome, and the kit covers human chromosome at an average interval of 9.2 cm.
The genomic DNA prepared in Example 1 was used as a template, and PCR (GeneAmp PCR System 9700G, manufactured by ABI) was carried out using a linkage mapping set. Detection of the amplification product (fragment) was carried out using an ABI PRISM 3100 Genetic Analyzer (manufactured by ABI). The fluorescent primer set for typing includes primers labeled with three types of fluorescent dyes such as 6-FAM (blue), VIC (green) and NED (yellow), and therefore, even with fragments of the same size, three types of colors can be separately discriminated. Accordingly, large amounts of samples could be rapidly processed.
The typing of the fragments was carried out by means of Genotyper Software v3.7 (manufactured by ABI) and GeneScan Software (manufactured by ABI):
A statistical test of the linkage was carried out using Genehunter v2.1_r5 Software (Kruglyak, L. et al., Am. J. Hum. Genet., 58(6), 1347-1363, 1996), which is a non-parametric analysis. Determination of the region where linkage is recognized was carried out according to the guidelines of Lander and Kruglyak (Nat. Genet., 11(3), 241-247, 1995) as described below, based on the criteria for obtaining false positive linkage.
A linkage analysis came to be actively carried out over the entire genome through the guidelines of Lander and Kruglyak (polygenic diseases), but in a linkage analysis of individual genes, the determination of whether the gene function can be a cause of a disease, is also needed. However, in an analysis over the entire genome, since the gene function is not taken into consideration at that stage, determination criteria (threshold values) that are purely meaningful in terms of mathematical genetics are required. Thus, they have provided significant linkage criteria as shown in the following Table 5, according to simulation results.










TABLE 5









Suggestive Linkage
P < 7.4 × 10−4



(Criteria for obtaining one false positive
LOD > 2.2



linkage result over the entire genome)



Significant Linkage
P < 2.2 × 10−5



(Criteria for obtaining 0.05 false positive
LOD > 3.6



linkage results over the entire genome)



High Significant Linkage
P < 3.0 × 10−7



(Criteria for obtaining 0.01 false positive
LOD > 5.4



linkage results over the entire genome)










As a result of the screening of whole chromosome, linkages were recognized on chromosome 1 and chromosome 11. The results are respectively presented in FIG. 2 and FIG. 3. As shown in FIG. 2, in chromosome 1, a maximum LOD score of 3.49 was obtained in the 1q21 to 1q23.1 region (near D1S498), and a maximum LOD score of 3.13 was obtained in the 1q32 to 1q41 region (D1S249-D1S213). As shown in FIG. 3, in chromosome 11, a maximum LOD score of 2.78 was obtained in the 11q12 to 11q13.5 region (D11S905 to D11S937). The values thus obtained satisfied the criteria of Suggestive Linkage defined by Lander and Kruglyak. Therefore, the curly hair trait locus could be specified on chromosome 1 and chromosome 11, and it was strongly suggested that hair shape susceptibility genes exist in these regions.
Example 3
Detailed Mapping in Candidate Regions
Subsequently, chromosome 1 where linkages was recognized in Example 2 was subjected to an affected sib-pair linkage analysis (detailed mapping), by further using microsatellite markers, for the purpose of narrowing the linkage regions.
The microsatellites used as a marker for the detailed mapping, were searched using Comprehensive human genetic maps of the Mammalian Genotyping Service (http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp). Microsatellites which were present in the genome at an interval of 1 to 2 cM and had high heterozygosity were selected. Furthermore, the fluorescent primers for typing, which were intended to amplify the microsatellites, were designed based on the Genome Database Project (GDB) (http://www.gdb.org/). Here, although the GDB has terminated the operation, currently retrieval and design can be carried out through the NCBI (http://www.ncbi.nlm.nih.gov/). Fluorescent primers for typing manufactured by ABI were used, and for some of the fluorescent primers for typing, those included in a linkage mapping set (ABI PRISM Linkage Mapping Set-HD 5 v2.5, manufactured by ABI) were used. The microsatellites used as the markers for detailed mapping, and the fluorescent primers for typing are presented in Table 6-1 and Table 6-2 (see SEQ ID NO:4 to NO:41).







TABLE 6-1







Microsatellites used as markers for detailed mapping, and fluorescent primers for typing




















Amplifi-








Gen-

cation







Loca-
Bank
Hetero-
product







tion
Acces-
zygos-
(fragment)





ABI
Microsatellite
(cM)
sion
ity
size
Label
Forward primer
Reverse primer



















MD10
AFM249zg9
D1S252
150.27
Z17138
0.82
 99-119






GATA12A07
D1S534
151.88
G07791
0.83
196-212
VIC
AGCACATAGCAGGCACTAGC
CGATTGTGCCACTACACAGT










(SEQ ID NO: 4)
(SEQ ID NO: 5)



AFMa297xg9
D1S2696
153.59
Z52819
0.88
159-185
6-FAM
AAAAATGAGTCCAGTAGAAGCCT
AGCCAGATTTACATCCCAG










(SEQ ID NO: 6)
(SEQ ID NO: 7)


 


MD10
AFM336xb1
D1S498
155.89
Z24441
0.82
183-205






AFM207yh6
D1S2346
158.75
Z51162
0.83
 89-115
VIC
TATCTTGCCCTGCACC
AAGTGGGTCTCCCCAG










(SEQ ID NO: 8)
(SEQ ID NO: 9)



AFMb009zb9
D1S2721
161.05
Z53073
0.74
233-247
VIC
TTGCTCGGCCAGAGTCT
ACGCATCACACCTGGCTAGT










(SEQ ID NO: 10)
(SEQ ID NO: 11)



AFMa127wh9
D1S506
163.34
Z24627
0.58
123-141
VIC
GGGCCTATGGCTGGAA
GGCTATGCTGGGGCAA










(SEQ ID NO: 12)
(SEQ ID NO: 13)


 


HD5
AFMa133ye5
D1S2635
165.62
Z52215
0.86
142-159






AFMb334xb1
D1S2771
168.52
Z53685
0.72
243-259
6-FAM
TCAGTTCCATAGGCTGACG
CATTGCTGATGCTGGAGG










(SEQ ID NO: 14)
(SEQ ID NO: 15)


 


MD10
AFM297wb9
D1S484
169.68
Z24182
0.64
136-142





 


MD10
AFMa057ze5
D1S2878
177.86
Z51743
0.84
169-195






AFMb316zb9
D1S2762
179.10
Z53529
0.81
232-250
NED
CCTTAATTGTGGTGTTGGT
AAAAATCTGGAAGGCATAAA










(SEQ ID NO: 16)
(SEQ ID NO: 17)


 


MD10
AFM063xg9
D1S196
181.49
Z16503
0.73
267-279






AFMb359xf5
D1S2799
183.19
Z53881
0.87
191-209
6-FAM
AGCAAGACCCTGTCTCAAAA
TGGATAGCTTTCCACCACT










(SEQ ID NO: 18)
(SEQ ID NO: 19)


 


HD5
AFM248wg5
D1S452
188.85
Z23809
0.76
119-131





 


MD10
AFM157xe7
D1S218
191.52
Z16701
0.83
266-286






AFM123yc5
D1S460
194.32
Z23379
0.84
145-159
6-FAM
ACAAGGTGACCGGAAAGACC
AGCTCTGGCAAGTTGAAGGA










(SEQ ID NO: 20)
(SEQ ID NO: 21)


 


HD5
AFMc025xh9
D1S2818
198.30
Z54047
0.70
258-268






AFM348tg1
D1S2848
200.96
Z51502
0.82
105-123
 VIC
ATCTGGGTTCACTATTAAACAGAGT
TGGGCAAGGTAGAATATGTG










(SEQ ID NO: 22)
(SEQ ID NO: 23)


 


MD10
AFM205xg1
D1S238
202.73
Z16920
0.86
272-302





 


HD5
AFMa057vb5
D1S2877
205.40
Z51735
0.72
143-157





 


HD5
AFM031xd12
D1S412
209.15
Z23298
0.71
129-147





 


MD10
AFM165xc9
D1S413
212.44
Z23420
0.77
246-262






UT492
D1S373
214.08
L16266
0.90
283-330
VIC
GGGTGACAGAGCAAGACTC
CCCTGACCTCCCTTACAGA










(SEQ ID NO: 24)
(SEQ ID NO: 25)



AFM136xa7
D1S1723
215.17
Z51003
0.83
167-181
NED
AACTGTGTCCAGCAGCAACT
TATGTGCCTGTTGTGTGCAT










(SEQ ID NO: 26)
(SEQ ID NO: 27)



AFMa190xd5
D1S2655
216.82
Z52412
0.90
224-260
VIC
AGGGTCCCCAAAGAGCCTTC
ATGGCAGCACATCCTGCTTC










(SEQ ID NO: 28)
(SEQ ID NO: 29)



AFMa224xc1
D1S2668
218.46
Z52594
0.77
233-247
VIC
AATCACTTGAACCTGGGAG
ACTGACTGGCTGTTTCTGAG










(SEQ ID NO: 30)
(SEQ ID NO: 31)
























TABLE 6-2










Amplifi-








Gen-

cation







Loca-
Bank
Hetero-
product







tion
Acces-
zygos-
(fragment)





ABI
Microsatellite
(cM)
sion
ity
size
Label
Forward primer
Reverse primer
























MD10
AFM234wf6
D1S249
220.65
Z17051
0.87
155-185





 


HD5
AFMa290xd1
D1S2692
222.84
Z52805
0.87
276-316






AFMa082wf9
D1S2891
224.50
Z51920
0.75
211-273
6-FAM
ACTGCTTATTCGGAGTTGGA
CCAAGAGTTTTCTTAGCAAATC










(SEQ ID NO: 32)
AC











(SEQ ID NO: 33)


 


HD5
AFM224xc1
D1S245
227.81
Z17011
0.83
239-257






AFM108ya3
D1S205
229.13
Z16585
0.80
 94-112
6-FAM
CTGAGCACAGCAGTGGTCTC
AAGGCTTATCAAGAGCGAGG










(SEQ ID NO: 34)
(SEQ ID NO: 35)


 


MD10
AFM203zb6
D1S425
231.11
Z23538
0.81
 92-108






GATA87F04
D1S2141
233.38
G07856
0.82
236-263
6-FAM
AGACTTACAGCACTGGCTGC
TGCTCCTAGGAAAGGAAACA










(SEQ ID NO: 36)
(SEQ ID NO: 37)



AFM297xc1
D1S2827
234.52
Z51306
0.78
142-152
6-FAM
GCTTCTGGCCTCTGTCA
AATTTTGCGTGTGTGTGC










(SEQ ID NO: 38)
(SEQ ID NO: 39)


 


HD5
AFM184yf6
D1S227
238.52
Z16806
0.71
 61-75






AFMa052zd1
D1S2871
241.26
Z51685
0.84
215-241
NED
TGAAGTGTGCATTCTNTACAT
CGAGACATTTGCATCATCA










CA
(SEQ ID NO: 41)










(SEQ ID NO: 40)



 


MD10
AFM147xf8
D1S213
242.34
Z16668
0.86
104-124









The results obtained by carrying out an affected sib-pair linkage analysis (detailed mapping) on chromosome 1 in the same manner as in Example 2, are presented in FIG. 4. As shown in FIG. 4, a maximum LOD score of 3.60 was obtained in the 1q21.3 region (D1S2696-D1S2346), and a maximum LOD score of 2.14 was obtained in the 1q32.1 to 1q32.2 region (D1S249 to D1S2891). The values thus obtained were considered to satisfy the criteria of Significant Linkage and Suggestive Linkage, respectively, defined by Lander and Kruglyak as described in Example 2. Therefore, the curly hair trait loci on chromosome 1 could be narrowed, and it was strongly suggested that hair shape susceptibility genes exist in these regions.
Example 4
Case-Control Association Analysis
In order to identify a hair shape susceptibility gene from the 1q32.1 to 1q32.2 region (D1S249 to D1S2891) on chromosome 1, where strong linkage was recognized in Example 3 above, a comparison of the allele frequency for the single nucleotide polymorphism (SNP) markers present in the region was made by a case-control association analysis.
Since it is necessary that the cases (affected: those having the curly hair trait) and the controls (control: those having the straight hair trait) consist of people of the same race as the race for whom the hair shape susceptibility gene is identified, in the present invention, non-family related Japanese people having the curly hair trait and non-family related Japanese people having the straight hair trait were employed as objects. Objects were collected in the same manner according to the criteria described in Example 1, and genomic DNA was obtained from each of 43 non-family related Japanese people having the curly hair trait and 51 non-family related Japanese people having the straight hair trait.
With reference was made to the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) and the JSNP database (http://snp.ims.u-tokyo.ac.jp/index_ja.html), SNPs which represented certain regions in the region to be analyzed, and had a gene frequency of the minor allele of 10% or higher in a panel of Japanese people, were selected as SNPs to be typed. Thus, 57 SNPs were selected from the region to be analyzed.
The typing of SNPs was carried out according to a TaqMan PCR method, using TaqMan SNP Genotyping Assays (manufactured by ABI, formerly known as Assays-on-Demand or Assays-by-Design). Furthermore, the apparatuses of Applied Biosystems 7900HT Fast Real-time PCR System (manufactured by ABI) and Applied Biosystems 7500 Real-time PCR System (manufactured by ABI) were used. The method was carried out according to the respective manuals attached to the apparatuses.
The typing data thus obtained were totalized for each of the cases and the controls, and a significant difference test was carried out through χ2 test by four methods involving the genotype, allele type, dominant model and recessive model. That is, if any genetic variation is causative of changes in the hair shape, differences in the allele frequency and the like are expected between the cases and the controls. Furthermore, in the present Example, since the association analysis was carried out on a relatively small number of objects, the significance level was set at p<0.05. Further, in some part, the significance level was set to be loose (p<0.1) in order to increase the power of the test.
As a result, it was found that there is a statistically significant (p<0.05) difference between the cases and the controls.
In SNP: rs1495840 (single nucleotide polymorphism represented by Nucleotide Number 50038 in the base sequence set forth in SEQ ID NO:2), the proportion of homozygous T-allele carriers was significantly higher in the people having the straight hair trait as compared with the people having the curly hair trait (Table 7-1), and even by the allele type, a significant difference was observed between the people having the straight hair trait and the people having the curly hair trait (Table 7-1).
Furthermore, it was found that even the two SNPs shown below exhibit a difference between the cases and the controls.
In SNP:rs576697 (single nucleotide polymorphism represented by Nucleotide Number 1 in the base sequence set forth in SEQ ID NO:1), the proportion of homozygous C-allele carriers was higher in the people having the curly hair trait as compared with the people having the straight hair trait (p=0.096) (Table 7-2).
In SNP:rs823114 (single nucleotide polymorphism represented by Nucleotide Number 60701 in the base sequence set forth in SEQ ID NO:3), the proportion of homozygous A-allele carriers was higher in the people having the curly hair trait as compared with the people having the straight hair trait (p=0.065) (Table 7-3).
These three SNPs all satisfied the Hardy-Weinberg equilibrium. Therefore, these three SNPs were determined to be hair shape susceptibility SNPs, and their relations with hair shape were confirmed.







TABLE 7-1





Association analysis on SNP: rs1495840

















SNP: rs1495840










Allele type
Genotype













A
T
AA
AT
TT





Curly hair trait
21.4%
78.6%
2.4%
38.1%
59.5%


Straight hair trait
10.8%
89.2%
0.0%
26.1%
78.4%


(control)














p value
Allele type
0.046



(χ2 test)
Genotype
0.102




AA vs AT, TT
0.048

















TABLE 7-2





Association analysis on SNP: rs576697

















SNP: rs576697










Allele type
Genotype













T
C
TT
TC
CC





Curly hair trait
14.0%
86.0%
2.3%
23.3%
74.4%


Straight hair trait
23.5%
76.5%
5.9%
35.3%
58.8%


(control)














p value
Allele type
0.096



(χ2 test)
Genotype
0.261




TT, TC vs CC
0.112

















TABLE 7-3





Association analysis on SNP: rs823114

















SNP: rs823114










Allele type
Genotype













G
A
GG
GA
AA





Curly hair trait
40.7%
59.3%
18.6%
44.2%
37.2%


Straight hair trait
52.0%
48.0%
24.0%
56.0%
20.0%


(control)














p value
Allele type
0.123



(χ2 test)
Genotype
0.183




GG vs GT, TT
0.065










Example 5
Haplotype Analysis
As a result of the analyses in Example 4, three hair shape susceptibility SNPs were found. Further, a haplotype analysis was carried out in order to found a correlation between hair shape and polymorphiosms that are present in the surrounding regions of the SNPs, particularly those that have not been typed, and to identify hair shape susceptibility genes.
In the analysis, the linkage disequilibrium coefficient D′ (pair-wise LD coefficient) based on the EM algorithm was calculated using Haploview 4.1 Software (Barrett, J C, et al., Bioinformatics, 21 (2), 263-265, 2005), and the analysis was carried out. A linkage disequilibrium analysis was carried out on the SNPs found above and the SNPs present in the surrounding regions, using the HapMap PHASE data of the International HapMap Project Database (HapMap Data ReI 21/PhaseII July 6, on NCBI Build 35 assembly, dbSNP b125). Meanwhile, the analysis panel consisted of JPT+CHB (Japanese people in Tokyo, Japan, and Chinese people of Han race in Beijing, China).
The method for inferring the haplotype block used the confidence interval (Gabriel, S B, et al., Science, 296 (5576), p. 2225-2229, 2002). That is, it can be considered that the haplotype blocks to be determined are mostly in the genome range where historical recombination has not been recognized, and strong linkage disequilibrium exists within the regions. Usually, when the upper limit of the 95% confidence interval of the linkage disequilibrium coefficient D′ is lower than 0.9, the region is considered as a region having an evidence of historical recombination. On the other hand, when the upper limit of the 95% confidence interval of D′ is higher than 0.98 and the lower limit is higher than 0.7, the region can be considered as a region where strong linkage disequilibrium exists.
As a result, haplotype blocks of the following items (1) to (3) containing the three hair shape susceptibility SNPs shown below were found.
(1) A 3,926-bp haplotype block ranging from SNP:rs576697 to SNP:rs12403361 containing SNP:rs576697 and represented by the base sequence set forth in SEQ ID NO:1 (FIG. 5). This haplotype block was a region containing CSRP1 gene. From this result, CSRP1 gene was identified as a hair shape susceptibility gene.
(2) A 76,945-bp haplotype block ranging from SNP:rs2820290 to SNP:rs2250377 containing SNP:rs1495840 and represented by the base sequence set forth in SEQ ID NO: 2 (FIG. 6). This haplotype block was a region containing NAV1 gene, IPO9 gene, and TMEM58 gene. From this result, NAV1 gene, IPO9 gene, and TMEM58 gene were identified as hair shape susceptibility gene.
(3) A 68,637-bp haplotype block ranging from SNP:rs823103 to SNP:rs1772150 containing SNP:rs823114 and represented by the base sequence set forth in SEQ ID NO:3 (FIG. 7). This haplotype block was a region containing NUCKS1 gene. From this result, NUCKS1 gene was identified as a hair shape susceptibility gene.
Example 6
Identification of Hair Shape Susceptibility SNP Marker
While haplotype blocks were found in the haplotype analysis in Example 5, a haplotype was extracted from each of the haplotype blocks using the same Haploview 4.1 Software (Barrett, J C et al., Bioinformatics, 21(2), 263-265, 2005). By comparing the respective nucleotide combinations of the extracted haplotypes, that is, the SNP marker groups, SNP loci that were linked to the hair shape susceptibility SNP marker loci were identified. The SNP loci thus identified can be identified as additional hair shape susceptibility SNP markers.
As a result, additional hair shape susceptibility SNP markers shown below were respectively found in the haplotype blocks of (1) to (3) shown in Example 4.
(1) 3,926-bp haplotype block represented by the base sequence set forth in SEQ ID NO:1: There were seven principal haplotypes in this haplotype block (Table 8). As the SNP loci that are linked to a hair shape susceptibility SNP marker, SNP:rs576697, additional three hair shape susceptibility SNP markers shown below were identified.
SNP:rs645390 (single nucleotide polymorphism represented by Nucleotide Number 1635 in the base sequence set forth in SEQ ID NO:1), SNP:rs3767542 (single nucleotide polymorphism represented by Nucleotide Number 2527), and SNP:rs675508 (single nucleotide polymorphism represented by Nucleotide Number 3766).











TABLE 8









Nucleotide number

Hair shape



in base sequence set
Haplotype
susceptibility
















SNP marker
forth in SEQ ID NO: 1
1
2
3
4
5
6
7
SNP



















rs576697
1
T
T
C
T
T
T
T
◯ (Example 4)


rs645390
1635
G
G
A
G
G
G
G
◯


rs4915528
2491
C
C
C
C
C
A
C


rs3767542
2527
G
A
A
A
A
G
A
◯


rs3767541
2622
C
C
C
T
C
C
C


rs12729389
3511
G
G
G
T
G
G
G


rs675508
3766
C
C
A
C
C
C
A
◯


rs12403361
3926
T
A
T
A
T
T
T









(2) 76,945-bp haplotype block represented by the base sequence set forth in SEQ ID NO:2: There were ten principal haplotypes in this haplotype block (Table 9). As SNP loci that are linked to a hair shape susceptibility SNP marker, SNP:rs1495840, additional 5 hair shape susceptibility SNP markers shown below were identified.
SNP:rs2271763 (single nucleotide polymorphism represented by Nucleotide Number 7519 in the base sequence set forth in SEQ ID NO:2), SNP:rs10920260 (single nucleotide polymorphism represented by Nucleotide Number 16901), SNP:rs16849387 (single nucleotide polymorphism represented by Nucleotide Number 30270), SNP:rs12127375 (single nucleotide polymorphism represented by Nucleotide Number 31333), and SNP:rs10920269 (single nucleotide polymorphism represented by Nucleotide Number 63008).











TABLE 9









Nucleotide number

Hair shape



in base sequence set
Haplotype
susceptibility



















SNP marker
forth in SEQ ID NO: 2
1
2
3
4
5
6
7
8
9
10
SNP






















rs2820290
1
A
A
G
A
A
G
A
G
A
A



rs2820292
606
A
A
C
A
A
C
A
C
A
A


rs1022361
6589
G
A
A
G
A
A
A
A
A
A


rs1032524
7145
T
T
C
T
T
C
T
C
T
T


rs2271763
7519
G
G
G
G
G
A
G
A
G
G
◯


rs2644128
9759
C
C
G
C
C
G
C
G
C
C


rs10920259
14015
C
T
T
C
T
C
T
C
T
T


rs4950794
16645
T
A
T
T
A
T
T
T
A
A


rs10920260
16901
T
T
T
T
T
G
T
G
T
T
◯


rs2820295
17187
G
G
A
G
G
G
G
G
G
G


rs2644112
22425
T
T
C
T
T
T
T
T
T
T


rs2644119
24095
C
C
T
C
C
C
C
C
C
C


rs2644122
26726
A
A
G
A
A
A
A
A
A
A


rs12567555
28279
G
G
G
G
A
G
G
G
A
G


rs16849387
30270
A
A
A
A
A
G
A
G
A
A
◯


rs6701026
30323
C
C
T
C
C
T
T
T
C
C


rs12562614
31313
A
G
A
G
G
A
A
A
G
G


rs12127375
31333
C
C
C
C
C
G
C
G
C
C
◯


rs12042456
32891
A
G
G
G
G
G
G
G
G
G


rs12722743
33262
C
C
C
C
C
T
C
C
C
C


rs2644107
34948
T
T
C
T
T
T
T
T
T
T


rs1400875
37762
T
T
C
T
T
T
T
T
T
T


rs2172935
42659
C
C
T
C
C
C
C
C
C
C


rs1495840
50038
T
T
T
T
T
A
T
A
T
T
◯ (Example 4)


rs950114
56529
C
T
T
T
T
T
T
T
T
T


rs2820311
57795
A
A
G
A
A
A
A
A
A
A


rs2271764
60376
T
T
T
T
T
T
T
T
C
T


rs1043823
61751
C
C
C
C
C
C
T
C
C
C


rs8024
61894
C
C
A
C
C
C
C
C
C
C


rs10920269
63008
G
G
G
G
G
T
G
T
G
G
◯


rs12032537
64678


rs6427922
71731
G
G
G
G
G
A
G
A
G
A


rs10920270
72840
C
C
C
G
C
C
C
C
C
C


rs2250377
76945
G
G
A
G
G
G
G
G
G
G









(3) 68,637-bp haplotype block represented by the base sequence set forth in SEQ ID NO:3: There were seven principal haplotypes in this haplotype block (Table 10). As SNP loci that are linked to a hair shape susceptibility SNP markers, SNP:rs823114, additional one hair shape susceptibility SNP markers shown below were identified.
SNP:rs3805 (single nucleotide polymorphism represented by Nucleotide Number 24524 in the base sequence set forth in SEQ ID NO:3).











TABLE 10









Nucleotide number

Hair shape



in base sequence set
Haplotype
susceptibility
















SNP marker
forth in SEQ ID NO: 3
1
2
3
4
5
6
7
SNP



















rs823103
1
G
A
G
A
A
A
G



rs1172199
108
C
T
C
T
T
T
C


rs12132270
2109
T
C
C
C
C
C
C


rs1891091
2587
T
C
C
C
C
C
C


rs12752037
2783
C
A
C
A
A
A
C


rs10751444
3313
C
T
T
T
T
T
T


rs1172198
3887


rs6676110
4146
A
G
G
G
G
G
G


rs12118655
4647
G
A
A
A
A
A
A


rs6673687
11538
A
T
A
T
T
A
A


rs12748961
17432
C
T
T
T
T
C
T


rs12030754
19212
G
C
G
C
G
G
C


rs16856186
19295


rs3805
24524
G
T
T
T
G
G
T
◯


rs10900522
25236
T
C
T
C
T
T
C


rs951366
26521
T
C
T
C
T
T
C


rs823092
29042
T
T
A
T
T
T
T


rs823093
30396
A
A
G
A
A
A
A


rs11240557
33183


rs823108
34770
C
C
T
C
C
C
C


rs3761919
35889
G
A
G
A
G
G
A


rs1772146
37690
T
T
G
T
T
T
T


rs1772147
37840
A
A
G
A
A
A
A


rs1620334
39014
T
T
C
T
T
T
T


rs7513645
39195
G
A
G
A
G
G
A


rs823113
52406
G
G
C
G
G
G
G


rs823128
54547
A
A
G
A
A
A
A


rs2298143
57838


rs823114
60701
A
G
G
G
A
A
G
◯ (Example 4)


rs823117
64682
A
A
T
A
A
A
A


rs2096078
64947
G
G
G
A
G
G
A


rs823122
66197
T
T
C
T
T
T
T


rs823123
66515
T
T
C
T
T
T
T


rs1626710
68635
A
A
C
A
A
A
A


rs1772150
68637
A
A
G
A
A
A
A









Example 7
Analysis of Gene Expression in Scalp Hair Roots in Curly Hair People and Straight Hair People
Ten curly hair people and ten straight hair people were collected according to the classifications of Example 1, and an analysis was carried out on the expression of the hair shape susceptibility gene in the scalp hair roots of each test subject. In regard to the collection of specimens from the test subjects, an approval was granted in advance by the ethics committee, subsequently the person in charge of the implementation of informed consent explained the contents of the study to the objects using a written explanation, and written consent was obtained.
About 60 scalp hair strands per person were pulled out from all over the whole head of each test subject, and only those scalp hair root parts that were determined to be in the growth period from the shape of the hair root part, were collected in a petri dish filled with ice-cooled PBS (manufactured by Invitrogen, Inc.). Under a stereoscopic microscope and using forceps and a needle teeth, the outer hair root sheath and the inner hair root sheath were removed from the hair root part as much as possible, and the hair root of the hair shaft only (hair shaft keratinized region) was separated and prepared. The hair shaft keratinized region was introduced in a 1.5-mL tube containing 0.5 mL of an RNA extraction solution, ISOGEN (manufactured by Nippon Gene Co., Ltd.), and the tissue was sufficiently crushed with a mini codeless grinder and a homogenization pestle. 0.5 mL of ISOGEN and 200 μL of chloroform were added thereto, and the mixture was sufficiently stirred in a vortex mixer and then was centrifuged (15000 rpm, for 15 minutes) using a small-sized microcentrifuge. Thus, about 500 μL of an aqueous phase containing RNA was collected. 50 μL of 3 M sodium acetate and 1 μL of Ethachinmate (manufactured by Nippon Gene Co., Ltd.) were added to the collected solution, and the mixture was sufficiently stirred. Furthermore, 1 mL of isopropanol was added and stirred, and the mixture was centrifuged (15000 rpm, for 20 minutes) with a small-sized microcentrifuge to precipitate total RNA. The supernatant was discarded, and then 75% ethanol was added to the precipitate. The mixture was centrifuged again (15000 rpm, for 10 minutes) with a small-sized microcentrifuge. The supernatant was discarded, and the precipitate was dried in air and was dissolved in 20 μL of Nuclease-free Water (manufactured by Invitrogen, Inc.). A portion of this was used to measure the RNA concentration using an absorption spectrometer (GeneQuant: manufactured by Pharmacia AB, or NonoDrop: manufactured by Nanodrop Technologies, Inc.), or RiboGreen RNA Reagent and Kit (manufactured by Invitrogen, Inc.). cDNA was synthesized from 1 μg of the total RNA thus obtained using QuantiTect Reverse Transcription Kit (manufactured by Qiagen N.V.) according to the attached protocol, and the cDNA was used in the quantification of the amount of gene expression by PCR.
The quantification of the amount of gene expression was carried out using TaqMan (registered trademark) Gene Expression Assays manufactured by Applied Biosystems, Inc. (ABI). According to the attached protocol, the synthesized cDNA, a primer & probe set specific to the gene to be detected and quantified, a real-time PCR reagent and the like (manufactured by ABI) were mixed, and fragments of the gene to be detected and quantified were amplified with Applied Biosystems 7500 Real-Time PCR System (manufactured by ABI). At this time, real-time PCR was carried out in the same manner using a known cDNA derived from an standard hair shaft keratinized region sample, and a calibration curve was produced. Thus, standardization of the amount of gene expression was carried out. Furthermore, standardization of the amount of expression of the gene to be detected and quantified was carried out using GAPDH gene as an internal standard, and also employing KRT31 gene and KRT85 gene, which is recognized to be uniformly expressed in the sample hair shaft keratinized region, as internal standards.
In order to detect and quantify the amount of expression of CSRP1 gene, Assay Number Hs00187916_m1 of TaqMan Gene Expression Assays (manufactured by ABI) was used as a specific primer & probe set.
In order to detect and quantify the amount of expression of IPO9 gene, Assay Number Hs00949771_m1 of TaqMan Gene Expression Assays (manufactured by ABI) was used as a specific primer & probe set.
In order to detect and quantify the amount of expression of NUCKS1 gene, Assay Number Hs00224144_m1 of TaqMan Gene Expression Assays (manufactured by ABI) was used as a specific primer & probe set.
The amounts of expression of the hair shape susceptibility genes in the scalp hair roots of the curly hair group and the straight hair group are presented in FIG. 8A to FIG. 8C. From the results shown in FIG. 8, decreases in the amount of expression of IPO9 gene and NUCKS1 gene were observed and an increase in the amount of expression of CSRP1 gene was observed in the curly hair group, as compared with the straight hair group. Therefore, it was made clear that CSRP1 gene, IPO9 gene and NUCKS1 are hair shape susceptibility genes serving as indicators for the evaluation of hair shape, and the measurement of the amounts of expression of these genes in the hair root area is valuable.
Example 8
Screening of Substance Regulating Amount of Expression of Hair Shape Susceptibility Gene
Normal human neonatal foreskin epidermal keratinocytes (KK-4009, manufactured by Kurabo Industries, Ltd.) were used in the screening. Normal human neonatal foreskin epidermal keratinocytes in a frozen state were melted, and then the cells were seeded in a 75-cm2 flask or a 25-cm2 flask at a density of 2500 cells/cm2. The cells were cultured in a serum-free medium for human keratinocyte culture (Defined Keratinocyte-SFM, manufactured by Invitrogen, Inc.) containing added supplements, under the conditions of 37° C. and a CO2 concentration of 5%. The cells were subcultured at the time point at which the cells reached a sub-confluent state, and the cells were seeded in a 6-well plate at a cell density of 2500 cells/cm2. At the time point at which the cells had reached a sub-confluent state (Day 0), the medium was exchanged to a serum-free medium for human keratinocyte culture containing no supplements, and the cells on Day 1 were used as the cells for screening.
To the medium (serum-free medium for human keratinocyte culture containing no supplements) for the cells for screening prepared as described above, a plant extract was added to a final concentration of 0.1% or 1%, and the cells were cultured for 24 hours under the conditions of 37° C. and a CO2 concentration of 5%. Furthermore, as control, 50% ethanol (control) was similarly added to a final concentration of 0.1% or 1%, and the cells were cultured.
After completion of the culture (Day 2), the medium was removed by suction, the cells were washed two times with PBS (manufactured by Invitrogen, Inc.), and then 1 mL per well of ISOGEN (manufactured by Nippon Gene Co., Ltd.) was added to the cells. The cells were sufficiently lysed and mixed through pipetting, and the solution was collected in a 1.5-mL tube. Total RNA was extracted by the same method as the method described in Example 7, and cDNA for use in the quantification of the amount of gene expression by PCR was obtained. The quantification of the amount of expression of the hair shape susceptibility gene was also carried out by the method described in Example 7.
In regard to the determination criteria for a substance that regulates the amount of expression of a gene, for example, if the amount of gene expression is higher by 10%, preferably 30%, and more preferably 50% or more, as compared with the control, the amount of expression is then said to be significantly high, and the test substance can be selected as an expression promoting agent for the hair shape susceptibility gene. Furthermore, for example, if the amount of gene expression is lower by 10%, preferably 30%, and more preferably 50% or more, as compared with the control, the amount of expression is then said to be significantly low, and the test substance can be selected as an expression suppressing agent for the hair shape susceptibility gene.
Approximately 700 kinds of plant extracts were evaluated by the screening system described above, and a search was made for substances that regulate the amount of expression of the hair shape susceptibility gene. As a result, expression promoting agents and expression suppressing agent for the genes were respectively found as indicated in Table 11.







TABLE 11







Substances that regulate the amounts of expression


of the hair shape susceptibility genes









Name of plant extract















Amount of CSRP1 gene




expression




(relative to control as 1)


Expression
Verbena officinalis
3.26


promoting
(whole plant extract)


agent
Solanum lyratum
2.61



(whole plant extract)



Eucommia ulmoides
2.34



(bark extract)


Expression
Amomum cardamomum
0.67


suppressing
(seed extract)


agent
Eupatorium perfoliatum
0.44



(leaf and spike extract)



Morun alba
0.32



(leaf extract




Amount of IPO9 gene




expression




(relative to control as 1)


Expression
Fraxinus americana
2.54


promoting
(bark extract)


agent
Aesculus hippocastanum
2.17



(bark extract)



Centipeda minima
1.83



(whole plant extract)


Expression
Corylus heterophylla
0.58


suppressing
(seed kernel extract)


agent
Zingiber officinale
0.48



(root extract)



Euonymus atropurpureus
0.32



(bark extract)




Amount of NUCKS1 gene




(relative to control as 1)


Expression
Hippophae rhamnoides
2.08


promoting
(fruit extract)


agent
Centipeda minima
1.96



(whole plant extract)



Beta vulgaris
1.66



vulgaris L. (whole



plant extract)


Expression
Swertia japonica
0.49


suppressing
(whole plant extract)


agent
Lappula squarrosa
0.34



(fruit extract)



Eriobotrya japonica
0.20



(leaf extract)









Reference Example
Relations Between Hair Shape and Form of Hair Follicle
In general, the hair shape varies with the human races, and the people of the Asian race relatively more frequently have straight hair, while the people of the African race mainly have kinky hair (or curled hair). A large proportion of the people of the Indo-European race have a trait of wavy hair (wave hair) which is intermediate of the two. As a feature related to such variation of hair shape, the form of the hair follicle at the hair root part may be mentioned. That is, if the form of the hair follicle is curved, the hair is curved, and if the form of the hair follicle is straight, the hair is straight (Thibaut, S. et al., Br. J. Dermatol., 152(4), p. 632-638, 2005).
In order to investigate the relations between the hair shape and the form of the hair follicle in more detail, tissue specimens of hair follicle were produced from the human scalp tissues of various races, and the form of the hair follicle was observed. Meanwhile, in regard to the collection of specimens from the test subjects, an approval was granted in advance by the ethics committee, subsequently the person in charge of the implementation of informed consent explained the contents of the study to the objects using a written explanation, and written consent was obtained. The collected hair follicles were frozen after being embedded in Tissue-Tek OCT Compound (manufactured by Miles Laboratories, Inc.), which is an embedding medium for frozen tissue section preparation, and frozen section specimens were produced according to a standard method. Subsequently, the specimens were subjected to HE staining, and were observed with a microscope.
FIG. 9 presents images of the hair follicle tissue of various human races. As can be seen from the results shown in FIG. 9, the hair follicle of an Asian person having straight hair was straight, while the hair follicle of a Caucasian person having wavy hair was bent only at the lowermost part of the hair root. Furthermore, in the case of an Afro-American having curled hair, it was found that the entire hair follicle tissue was curved. Therefore, it could be confirmed that the hair shape and the form of the hair follicle were closely related to each other.
Example 9
Evaluation of Form of Hair Follicle Through Culture of Human Hair Follicle Organ
As a method for evaluating the hair shape and the form of the hair follicle, an investigation was conducted on an evaluation method based on the culture of the human hair follicle organ. The scalp tissues of the temporal region or the occipital region of men and women in the age of 30's to 80's, which had been excised by cosmetic plastic surgery and became unnecessary, were obtained and used in the experiment. Meanwhile, in regard to the collection of specimens, an approval was granted in advance by the ethics committee, subsequently the surgeon explained the contents of the study to the objects using a written explanation, and written consent was obtained.
The human scalp tissue thus obtained was recovered in a petri dish filled with Williams' E medium (manufactured by Sigma-Aldrich Company) containing 1% of antibiotic/antifungal agents (manufactured by Invitrogen, Inc.). The hair follicles were aseptically isolated one by one under a stereoscopic microscope and using forceps and a scalpel or a needle teeth. The isolated hair follicles were separated from the epidermal tissue at the position of the lower part of the sebaceous gland, and any extra connective tissue, adipocytes and the like attached to the lower part of the hair follicle, were removed as much as possible. The isolated hair follicles thus prepared were transferred, one hair follicle per well, onto a 24-well plate to which Williams' E medium (manufactured by Sigma-Aldrich Company) containing 400 μL of 10 μg/mL insulin (manufactured by Invitrogen, Inc.), 40 ng/mL of hydrocortisone (manufactured by Sigma-Aldrich Company), 2 mM L-glutamine (manufactured by Invitrogen, Inc.), and 1% antibiotic/antifungal agents (manufactured by Invitrogen, Inc.) had been added, and culture was initiated. The culture was carried out in the manner of suspension culture, under the conditions of 37° C. and a CO2 concentration of 5%. Thereafter, the medium was exchanged at an interval of 2 to 3 days, and at the same time, photographs of the hair follicles were taken.
The photographs of the change in the form of the hair follicle during culturing days are presented in FIG. 10. The hair shaft in the hair follicle grew with the progress of the culture, and thereby elongated. Furthermore, along with the progress of the culture, it was observed that the hair follicle was straight (straight hair) after one day from the initiation of culture (Day 1), but the hair follicle (hair shaft) was gradually curved with the culturing days.
In order to quantify the degree of curvature of the hair follicle (hair shaft), the ratio of end-to-end distance was calculated. The ratio of end-to-end distance is one of the indices representing the degree of curl, and can be determined by the following calculation (Hrdy, D., Am. J. Phys. Anthropol., 39(1), p. 7-17, 1973).
Straight Length Between the Ends of the Object (Hair, Hair Follicle)/Curve Length Along the Axis of the Object (Hair or Hair Follicle)
That is, according to the formula shown above, the ratio of end-to-end distance represents a value between 0 and 1, so that a straight object gives a value close to 1, and an object with a large degree of curvature gives a value close to zero (0).
The photographs of the hair follicles shown in FIG. 10 were analyzed using an image analyzing software (Nexus NewQube Ver. 4.23, manufactured by IMAX Systems, Inc.), and the length of the hair follicle (hair shaft) and the ratio of end-to-end distance were determined (Table 12).
As a result, it could be confirmed that the hair follicle (hair shaft) elongated with the culturing days, and at the same time, the hair follicle was gradually being curved. Therefore, it was found that when this evaluation system is used, search for an agent for curling of hair, or a curly hair ameliorating agent (hair straightening agent) can be conducted. That is, a test substance is added to the evaluation system of human hair follicle organ culture, the hair follicle organ is cultured, and the ratio of end-to-end distance of the hair follicle (hair shaft) which has elongated to a certain length is measured. When the hair follicle is cultured in the presence of a test substance, if the ratio of end-to-end distance becomes smaller as compared with a control cultured without adding the test substance, the test substance can be selected as a hair curling agent. When the hair follicle is cultured in the presence of a test substance, if the ratio of end-to-end distance becomes larger as compared with a control cultured without adding the test substance, the test substance can be selected as a curly hair ameliorating agent (hair straightening agent).







TABLE 12







Changes in the length of hair follicle (hair shaft) and the ratio


of end-to-end distance in the hair follicle during culturing











Ratio of


Culturing days
Length of hair
end-to-end


(day)
follicle (mm)
distance












1
3.465
1.005


3
4.419
1.002


6
5.732
0.997


8
6.748
0.988


10
7.571
0.973


12
8.131
0.958


14
8.758
0.901


16
9.433
0.825


18
9.720
0.818









Example 10
Evaluation of an Agent of Regulating the Expression of in Human Hair Follicle Organ Culture
For the purpose of verifying the effect of an agent of regulating the expression of hair shape susceptibility gene on the form of the hair follicle, an evaluation was conducted using the evaluation system for human hair follicle organ culture.
The human hair follicle was prepared according to Example 9. The isolated hair follicles were divided into two groups, with 12 hair strands in each group, so that there was no fluctuation in the size. One of the groups was suspension cultured for 15 days in a medium for organ culture (400 μL) to which a Centipede minima extract, which is an expression promoting agent for IPO9 gene and NUCKS1 gene as described in Table 11, was added at a final concentration of 0.2%. The other group was suspension cultured for 15 days in a medium for organ culture (400 μL) to which 50% EtOH (a final concentration of 0.83%) was added, as a control. According to the same procedure, a group added with an Amomum cardamomum extract (final concentration 0.2%), which is a CSRP1 gene expression suppressant as described in Table 11, and a control group (50% EtOH, final concentration 0.83%) were prepared (n=12 for each group).
After the initiation of culture, the medium was exchanged at an interval of 2 to 3 days, and at the same time, photographs of the hair follicles were taken. From the images of hair follicles thus taken, the degree of elongation and the degree of curvature (ratio of end-to-end distance) of the hair follicles were respectively measured.
At the time point at which the length of the hair follicle (hair shaft) elongated by 1.5 mm or more as compared with the length at the initiation of culture, the ratio of end-to-end distance of the hair follicle (hair shaft) was measured. As a result, it was found that the Centipeda minima extract and the Amomum cardamomum extract significantly increase the ratio of end-to-end distance, which indicates the degree of curvature of the hair follicle (hair shaft), as compared with the 50% EtOH-added control (FIG. 11). From these results, it could be seen that an agent of regulating the expression of hair shape susceptibility gene can be selected as a curly hair ameliorating agent (hair straightening agent) for the hair.

## Claims

1. A method for evaluating or selecting a hair shape regulating agent, comprising the following steps (a) to (f):
(a) culturing cells in the presence of a test substance, wherein the cells are capable of expressing a human CSRP1 gene, or a protein encoded by the gene, and wherein the cells are selected from the group consisting of epidermal keratinocytes, human hair follicle tissue cells and hair root area-derived cells;
(b) measuring the amount of expression of the CSRP1 gene or the protein in the cells that were cultured in the presence of the test substance;
(c) selecting, based on the results of step (b), a test substance that decreases or increases the amount of expression of the gene or the protein, as compared to that in control cells cultured in the absence of the test substance;
(d) culturing a human hair follicle in organ culture in the presence of the test substance that is selected in step (c);
(e) determining the degree of curl of the hair follicle's hair shaft that occurs as a result of the culturing as compared to that of the hair shaft of a control hair follicle that is cultured in the absence of the test substance; and
(f) selecting a test substance from step (e) that both (i) increases or decreases the amount of expression of the gene or the protein in step (c) and (ii) alters the degree of curl of the hair follicle's hair shaft in step (e), as a hair shape regulating agent; wherein a test substance that increases the amount of expression and curl is selected as a hair curling agent and a substance that decreases the amount of expression and curl is selected as a hair straightening agent.

2. The method of claim 1, wherein the test substance increases the amount of expression of the gene or the protein.

3. The method of claim 1, wherein the test substance decreases the amount of expression of the gene or the protein.

4. The method of claim 1, wherein the CSRP1 gene encodes a CSRP1 protein having the amino acid sequence of SEQ ID NO: 43.

5. The method of claim 4, wherein the sequence of the CSRP1 gene is that of SEQ ID NO: 42.

6. The method of claim 1, wherein the cells in step (a) are epidermal keratinocytes.

7. The method of claim 1, wherein the cells in step (a) are human hair follicle tissue cells.

8. The method of claim 1, wherein the cells in step (a) are hair root area-derived cells.

9. The method according to claim 1, wherein step (f) is selecting a test substance that increases the amount of expression and curl as a hair curling agent.

10. The method according to claim 1, wherein step (f) is selecting a test substance that decreases the amount of expression and curl as a hair straightening agent.

11. A method for evaluating or selecting a hair shape regulating agent, comprising the following steps (a) to (g):
(a) introducing a fusion gene to cells, wherein, in the fusion gene, a human CSRP1 gene's regulatory region is linked to and controls expression of a reporter gene, and wherein the cells are capable of expressing an expression product of the reporter gene and are selected from the group consisting of epidermal keratinocytes, human hair follicle tissue cells and hair root area-derived cells;
(b) culturing the cells that contain the fusion gene of step (a) in the presence of a test substance;
(c) measuring the amount of expression of the expression product of the reporter gene in the cells cultured in the presence of the test substance;
(d) selecting, based on the results of step (c), a test substance that increases or decreases the amount of the expression product of the reporter gene, as compared to that in control cells cultured in the absence of the test substance;
(e) culturing a human hair follicle in organ culture in the presence of the test substance that is selected in step (d);
(f) determining the degree of curl of the hair follicle's hair shaft that occurs as a result of the culturing as compared to that of the hair shaft of a control hair follicle that is cultured in the absence of the test substance; and
(g) selecting a test substance from step (f) that both (i) increases or decreases the amount of expression of the protein encoded by the reporter gene in step (d) and (ii) alters the degree of curl of the hair follicle's hair shaft in step (f) as a hair shape regulating agent,
wherein a test substance that increases the amount of expression and curl is selected as a hair curling agent and a substance that decreases the amount of expression and curl of is selected as a hair straightening agent.

12. The method of claim 11, wherein the test substance increases the amount of expression of the reporter gene.

13. The method of claim 11, wherein the test substance decreases the amount of expression of the reporter gene.

14. The method of claim 11, wherein the sequence of the regulatory region of the CSRP1 gene is that of SEQ ID NO: 48.

15. The method of claim 11, wherein the cells in step (a) are epidermal keratinocytes.

16. The method of claim 11, wherein the cells in step (a) are human hair follicle tissue cells.

17. The method of claim 11, wherein the cells in step (a) are hair root area-derived cells.

18. The method according to claim 11, wherein step (g) is selecting a test substance that increases the amount of the expression product of the reporter gene and curl, as a hair curling agent.

19. The method according to claim 11, wherein step (g) is selecting a test substance that decreases the amount of the expression product of the reporter gene and curl, as a hair straightening agent.